Nucleic acid molecules and other molecules associated with soybean cyst nematode resistance

ABSTRACT

The present invention is in the field of soybean genetics. More specifically, the invention relates to nucleic acid molecules from regions the soybean genome, which are associated with soybean cyst nematode resistance. The invention also relates to proteins encoded by such nucleic acid molecules as well as antibodies capable of recognizing these proteins. The invention also relates to nucleic acid markers from regions the soybean genome, which are associated with soybean cyst nematode resistance. Moreover, the invention relates to uses of such molecules, including, transforming soybean cyst nematode resistant soybean with constructs containing nucleic acid molecules from regions the soybean genome, which are associated with soybean cyst nematode resistance. Furthermore, the invention relates to the use of such molecules in a plant breeding program.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/754,853, filed Jan. 5, 2001, which claims the benefit under 35 U.S.C.§119(e) of U.S. Application No. 60/174,880, filed Jan. 7, 2000. Thedisclosures of U.S. application Ser. Nos. 09/754,853 and 60/174,880 areboth herein incorporated by reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

A paper copy of the Sequence Listing and a computer readable form (CRF)of the sequence listing on diskette, containing the file named00330V2.TXT, which is 2,521,108 bytes in size (measured in Windows XP),and which was recorded on Jul. 27, 2001, are herein incorporated byreference.

FIELD OF THE INVENTION

The present invention is in the field of soybean genetics. Morespecifically, the invention relates to nucleic acid molecules fromregions of the soybean genome, which are associated with soybean cystnematode (SCN) resistance. The invention also relates to proteinsencoded by such nucleic acid molecules as well as antibodies capable ofrecognizing these proteins. The invention also relates to nucleic acidmarkers from regions of the soybean genome, which are associated withSCN resistance. Moreover, the invention relates to uses of suchmolecules, including, transforming SCN sensitive soybean with constructscontaining nucleic acid molecules from regions in the soybean genome,which are associated with SCN resistance. Furthermore, the inventionrelates to the use of such molecules in a plant breeding program.

BACKGROUND OF THE INVENTION

The soybean, Glycine max (L.) Merril (Glycine max or soybean), is one ofthe major economic crops grown worldwide as a primary source ofvegetable oil and protein (Sinclair and Backman, Compendium of SoybeanDiseases, 3^(rd) Ed. APS Press, St. Paul, Minn., p. 106. (1989)). Thegrowing demand for low cholesterol and high fiber diets has alsoincreased soybean's importance as a health food.

Prior to 1940, soybean cultivars were either direct releases ofintroductions brought from Asia or pure line selections from geneticallydiverse plant introductions. The soybean plant was primarily used as ahay crop in the early part of the 19th century. Only a few introductionswere large-seeded types useful for feed grain and oil production. Fromthe mid 1930's through the 1960's, gains in soybean seed yields wereachieved by changing the breeding method from evaluation and selectionof introduced germplasm to crossing elite by elite lines. The continuouscycle of cross hybridizing the elite strains selected from the progeniesof previous crosses resulted in the modern day cultivars.

Over 10,000 soybean strains have now been introduced into the UnitedStates since the early 1900's (Bernard et al., United States NationalGermplasm Collections. In: L. D. Hil (ed.), World Soybean Research, pp.286-289. Interstate Printers and Publ., Danville, Ill. (1976)). Alimited number of those introductions form the genetic base of cultivarsdeveloped from the hybridization and selection programs (Johnson andBernard, The Soybean, Norman Ed., Academic Press, N.Y., pp. 1-73(1963)). For example, in a survey conducted by Specht and Williams,Genetic Contributions, Fehr eds. American Soil Association, Wisconsin,pp. 49-73 (1984), for the 136 cultivars released from 1939 to 1989, only16 different introductions were the source of cytoplasm for 121 of that136. Certain soybean strains are sensitive to one or more pathogens. Oneeconomically important pathogen is SCN.

SCN accounts for roughly 40% of the total disease in soybean and canresult in significant yield losses (up to 90%). SCN is the mostdestructive pest of soybean to date and accounts for an estimated yieldloss of up to $809 million dollars annually. Currently, the most costeffective control measures are crop rotation and the use of host plantresistance. While breeders have successfully developed SCN resistantsoybean lines, breeding is both difficult and time consuming due to thecomplex and polygenic nature of resistance. The resistance is often racespecific and does not provide stability over time due to changing SCNpopulations in the field. In addition, many of the resistant soybeanvarieties carry a significant yield penalty when grown in the absence ofSCN.

SCN, Heterodera glycines Inchinohe, was identified on soybeans in theUnited States in 1954 at Castle Hayne, N.C. Winstead, et al., Plant Dis.Rep. 39:9-11 (1955). Since its discovery the SCN has been recognized asone of the most destructive pests in soybean. It has been reported innearly all states in which soybeans are grown, and it causes majorproduction problems in several states, being particularly destructive inthe Midwestern states. See generally: Caldwell, et al., Agron. J.52:635-636 (1960); Rao-Arelli and Anand, Crop. Sci. 28:650-652, (1988);Baltazar and Mansur, Soybean Genet. Newsl. 19:120-122 (1992); Concibido,et al., Crop. Sci., (1993). For example, sensitive soybean cultivars had5.7-35.8% lower seed yields than did resistant cultivars on SCN race-3infested sites in Iowa. (Niblack and Norton, Plant Dis. 76:943-948(1992)).

Shortly after the discovery of SCN in the United States, sources of SCNresistance were identified (Ross and Brim, Plant Dis. Rep. 41:923-924(1957)). Some lines such as Peking and Plant Introduction (PI) PI88788,were quickly incorporated into breeding programs. Peking became widelyused as a source of resistance due to its lack of agronomicallyundesirable traits, with Pickett as the first SCN resistant cultivarreleased (Brim and Ross, Crop Sci. 6:305 (1966)). The recognition thatcertain SCN resistant populations could overcome resistant cultivarslead to an extensive screen for additional sources of SCN resistance.PI88788 emerged as a popular source of race 3 and 4 resistance eventhough it had a cyst index greater than 10% (but less than 20%) againstrace 4, and Peking and its derivatives emerged as a popular source forraces 1 and 3. PI437654 was subsequently identified as having resistanceto all known races and its SCN resistance was backcrossed into Forrest.Currently there are more than 130 PIs known to have SCN resistance.

SCN race 3 is considered to be the prominent race in the Midwesternsoybean producing states. Considerable effort has been devoted to thegenetics and breeding for resistance to race 3. While both Peking andPI88788 are resistant to SCN race 3, classical genetics studies suggestthat they harbor different genes for race 3 resistance (Rao-Arelli andAnand, Crop Sci. 28:650-652 (1988)). Crosses between PI88788(R) andEssex(S) segregate 9(R): 55(S) in the F₂ population and 1(R): 26(Seg):37(S) families in the F₃ generation, suggesting that resistance to race3 in PI88788 is conditioned by one recessive and two dominant genes,where as Peking and PI90763 resistance is conditioned by one dominantand two recessive genes. Based on reciprocal crosses, Peking, Forrest,and PI90763 have genes in common for resistance to SCN race 3(Rao-Arelli and Anand, Crop Sci., 28:650-652 (1988)). A cross betweenPeking and PI88788 segregates 13(R):3(S) in the F₂ generation,indicating a major difference between the parents for race 3 resistance.Generation mean analysis based on four crosses between resistant andsensitive genotypes; A20 (R), Jack (R), Cordell (R) and A2234 (S),suggests that an additive genetic model is sufficient to explain most ofthe genetic variation of race 3 SCN resistance in each cross, while theanalysis of the pooled data indicates the presence of dominant effectsas well (Mansur, Carriquiry and Roa-Arelli, Crop Sci. 33:1249-1253(1993)). This analysis further indicates that race 3 resistance isprobably under the genetic control of three, but not more than fourgenes.

RFLP analysis of segregating populations between resistant and sensitivelines; PI209332 (R), PI90763 (R), PI88788 (R), Peking (R) and Evan (S),identified a major SCN resistance QTL (rhg1) which maps to linkage groupG (Concibido et al., Theor Appl. Genet. 93:234-241 (1996)). In thisstudy, rhg1 explains 51.4% of the phenotypic variation in PI209322,52.7% of the variation in PI90763, 40.0% of the variation in PI88788 and28.1% of the variation in Peking. This major resistance QTL was assumedbe one and the same in all of the mapping populations employed. However,as pointed out by the authors, it is possible that the genomic intervalcontains distinct but tightly linked QTLs. In a related study usingPI209332 as the source of resistance, Concibido et al., Crop Sci.36:1643-1650 (1996), show that a QTL on linkage group G (rhg1) iseffective against the three SCN races tested, explaining 35% of thephenotypic variation to race 1, 50% of the variation to race 3, and 54%of the variation to race 6. In addition to the major QTL on linkagegroup G, 4 other QTLs mapping to linkage groups D, J, L and K wereidentified, with some of the resistance loci behaving in a race specificmanner.

Concibido et al. (Crop Sci. 37:258-264 (1997)) found significantassociation of marker C006V to a major QTL on linkage group G (rhg1) andresistance to race 1, race 3 and race 6, in Peking and PI90763 (Evan XPeking, Evan X PI90763) and races 3 and 6 in PI88788 (Evan X PI88788),in agreement with the previous study based on the P209332 source ofresistance (Concibido et al., Crop Sci. 36:1643-1650 (1996)). Theresistance locus near C006V was effective against all races tested inall of the resistance sources. While statistically significant againstall races, this locus accounts for different proportions of the totalphenotypic variation with the races tested. For example, in PI90763 theresistance locus near C006V explains more than three times thephenotypic variation against race 1 than against race 3. The variabilitycan be attributed to differences in the genetic backgrounds, variabilityamong the SCN populations or may be a reflection of the limited size ofthe plant populations which were employed. This study further identifiedthree additional independent SCN resistance QTLs; one near the RFLPmarker A378H mapping to the opposite end of linkage group G from C006V(rhg1), one near the marker B032V-1 on linkage group J and a thirdlinked to A280Hae-1 on linkage group N. Comparisons between thedifferent SCN races indicated that some of the putative SCN QTLs behavein a race specific manner.

PI437654 was identified as having resistance to all known races. Basedon analysis of 328 recombinant inbreed lines (RIL) derived from a crossbetween PI437654 and BSR101, Webb reported six QTLs associated with SCNresistance on linkage groups A2, C1, G, M, L25 and L26 (U.S. Pat. No.5,491,081). An allele on linkage group G, presumed to be rhg1, isinvolved with certain SCN races tested (races 1, 2, 3, 5 and 14), andhas the largest reported phenotypic effect on resistance to every race.In contrast, the QTLs on linkage groups A2, C1, M, L25 and L26 act in arace specific manner. The QTL on linkage group L25 was reportedlyinvolved with four of the five races, while the QTLs on linkage groups,A2, C1 and L26 were each involved in resistance to two of the five races(U.S. Pat. No. 5,491,081). Webb further reports data that the resistanceto any of the five races is likely to result from the combined effectsof the QTL involved in each race (U.S. Pat. No. 5,491,081).

Qui et al. (Theor Appl Genet 98:356-364 (1999)) screened 200 F_(2:3)families derived from a cross between Peking and Essex and identifiedRFLP markers which are associated with SCN resistance QTLs on linkagegroups B, E, I and H. The three QTLs on linkage groups B, E and Hjointly account for 57.7% of the phenotypic variation to race 1, theQTLs on linkage groups H and B account for 21.4% of the variation torace 3, while the QTLs on linkage groups I and E are associated withresistance to race 5 accounting for 14.0% of the phenotypic variation.In contrast to previous mapping studies which use Peking as the sourceof resistance, no significant association was detected to the rhg1 locuson linkage group G. The authors point out that the marker Bng122, whichhas been shown to have significant linkage to rhg1, is not polymorphicin the population employed (Concibido et al., Crop Sci. 36:1643-1650(1996)).

It has been reported that the rhg1 locus on linkage group G is necessaryfor the development of resistance to any of the SCN races. There havebeen efforts to develop molecular markers to identify breeding linesharboring the rhg1 SCN resistant allele. One of the most commonly usedmarkers for marker assisted selection (MAS) of rhg1 is an SSR locus thatco-segregates and maps roughly 0.4 cM from rhg1. This SRR marker,BARC-Satt_(—)309 is able to distinguish most, if not all, of the SCNsensitive genotypes from those harboring rhg1 from important sources ofresistance such as Peking and PI437654. Two simple sequence repeatmarkers have been reported that can be used to select for SCN resistanceat the rhg1 locus (Concibido et al., Theor Appl Genet 99: 811-818(1999)). Satt_(—)309 was also effective in distinguishing SCN resistantsources PI88788 and PI209332 in many, but not all, sensitive genotypes.In particular, Satt_(—)309 can not be used for MAS in populationsdeveloped from “typical” southern US cultivars (e.g., Lee, Bragg andEssex) crossed with resistance sources PI88788 or PI209332.

Matson and Williams have reported a dominant SCN resistance locus, Rhg4,which is tightly linked to the ‘i’ locus on linkage group A2 (Matson andWilliams, Crop Sci. 5:447 (1965)). The QTL reported by Webb on linkagegroup A2 maps near the ‘i’ locus and is considered to be Rhg4 (U.S. Pat.No. 5,491,081). Webb concludes that only two loci on linkage groups A2(Rhg4) and G (rhg1) explain the genetic variation to race 3.

SUMMARY OF THE INVENTION

The present invention includes and provides a method for the productionof a soybean plant having an rhg1 SCN resistant allele comprising: (A)crossing a first soybean plant having an rhg1 SCN resistant allele witha second soybean plant having an rhg1 SCN sensitive allele to produce asegregating population; (B) screening the segregating population for amember having an rhg1 SCN resistant allele with a first nucleic acidmolecule capable of specifically hybridizing to linkage group G, whereinthe first nucleic acid molecule specifically hybridizes to a secondnucleic acid molecule that is linked to the rhg1 SCN resistant allele;and, (C) selecting the member for further crossing and selection.

The present invention includes and provides a method of investigating anrhg1 haplotype of a soybean plant comprising: (A) isolating nucleic acidmolecules from the soybean plant; (B) determining the nucleic acidsequence of an rhg1 allele or part thereof; and, (C) comparing thenucleic acid sequence of the rhg1 allele or part thereof to a referencenucleic acid sequence. The present invention includes and provides amethod of introgressing SCN resistance or partial SCN resistance into asoybean plant comprising: performing marker assisted selection of thesoybean plant with a nucleic acid marker, wherein the nucleic acidmarker specifically hybridizes with a nucleic acid molecule having afirst nucleic acid sequence that is physically linked to a secondnucleic acid sequence that is located on linkage group G of soybeanA3244, wherein the second nucleic acid sequence is within 500 kb of athird nucleic acid sequence which is capable of specifically hybridizingwith the nucleic acid sequence of SEQ ID NO: 5, 6, complements thereof,or fragments thereof having at least 15 nucleotides; and, selecting thesoybean plant based on the marker assisted selection.

The present invention includes and provides a method for the productionof a soybean plant having an Rhg4 SCN resistant allele comprising: (A)crossing a first soybean plant having an Rhg4 SCN resistant allele witha second soybean plant having an Rhg4 SCN sensitive allele to produce asegregating population; (B) screening the segregating population for amember having an Rhg4 SCN resistant allele with a first nucleic acidmolecule capable of specifically hybridizing to linkage group A2,wherein the first nucleic acid molecule specifically hybridizes to asecond nucleic acid molecule linked to the Rhg4 SCN resistant allele;and, (C) selecting the member for further crossing and selection.

The present invention includes and provides a method of investigating anRhg4 haplotype of a soybean plant comprising: (A) isolating nucleic acidmolecules from the soybean plant; (B) determining the nucleic acidsequence of an Rhg4 allele or part thereof; and (C) comparing thenucleic acid sequence of the Rhg4 allele or part thereof to a referencenucleic acid sequence.

The present invention includes and provides a method of introgressingSCN resistance or partial SCN resistance into a soybean plantcomprising: performing marker assisted selection of the soybean plantwith a nucleic acid marker, wherein the nucleic acid marker specificallyhybridizes with a nucleic acid molecule having a first nucleic acidsequence that is physically linked to a second nucleic acid sequencethat is located on linkage group A2 of soybean A3244, wherein the secondnucleic acid sequence is within 500 kb of a third nucleic acid sequencewhich specifically hybridizes with the nucleic acid sequence of SEQ IDNO: 7, complements thereof, or fragments thereof having at least 15nucleotides; and, selecting the soybean plant based on the markerassisted selection.

The present invention includes and provides a substantially purifiednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 5, 6, 8-23, 28-43, complementsthereof, and fragments of either.

The present invention includes and provides a substantially purifiedfirst nucleic acid molecule with nucleic acid sequence whichspecifically hybridizes to a second nucleic acid molecule having anucleic acid sequence selected from the group consisting of a complementof SEQ ID NOs: 5, 6, 8-23, 28-43.

The present invention includes and provides a substantially purifiednucleic acid molecule comprising a nucleic acid sequence selected fromthe group consisting of SEQ ID NOs: 7, 44-47, and 50-53, complementsthereof, and fragments of either.

The present invention includes and provides a substantially purifiedfirst nucleic acid molecule with nucleic acid sequence whichspecifically hybridizes to a second nucleic acid molecule having anucleic acid sequence selected from the group consisting of a complementof SEQ ID NOs: 50-53.

The present invention includes and provides a substantially purifiedprotein or fragment thereof comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 1097, 1098, and 1100-1115 andfragments thereof.

The present invention includes and provides a substantially purifiedprotein or fragment thereof comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs 1099, and 1116-1119 andfragments thereof.

The present invention includes and provides a transformed plant having anucleic acid molecule which comprises: (A) an exogenous promoter regionwhich functions in a plant cell to cause the production of a mRNAmolecule; (B) a structural nucleic acid molecule encoding a protein orfragment thereof comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1097, 1100, 1098, 1101, 1102-1115; and(C) a 3′ non-translated sequence that functions in the plant cell tocause termination of transcription and addition of polyadenylatedribonucleotides to a 3′ end of the mRNA molecule.

The present invention includes and provides a transformed plant having anucleic acid molecule which comprises: (A) an exogenous promoter regionwhich functions in a plant cell to cause the production of a mRNAmolecule; (B) a structural nucleic acid molecule encoding a protein orfragment thereof comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 1099, 1116-1119; and (C) a 3′non-translated sequence that functions in the plant cell to causetermination of transcription and addition of polyadenylatedribonucleotides to a 3′ end of the mRNA molecule.

The present invention includes and provides a transgenic seed having anucleic acid molecule which comprises: (A) an exogenous promoter regionwhich functions to cause the production of a mRNA molecule; (B) astructural nucleic acid molecule encoding a protein or fragment thereofcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1097, 1100, 1098, 1101, 1102-1115; and (C) a 3′non-translated sequence that functions to cause termination oftranscription and addition of polyadenylated ribonucleotides to a 3′ endof the mRNA molecule.

The present invention includes and provides a transgenic seed having anucleic acid molecule which comprises: (A) an exogenous promoter regionwhich functions to cause the production of a mRNA molecule; (B) astructural nucleic acid molecule encoding a protein or fragment thereofcomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 1099, 1116-1119; and (C) a 3′ non-translated sequence thatfunctions to cause termination of transcription and addition ofpolyadenylated ribonucleotides to a 3′ end of the mRNA molecule.

DESCRIPTION OF THE FIGURES

FIG. 1 is an amino acid sequence alignment of the leucine rich repeatdomain of rhg1.

FIG. 2 is an amino acid sequence alignment of the leucine rich repeatdomain of Rhg4.

DESCRIPTION OF THE SEQUENCE LISTINGS

The following sequence listings form part of the present specificationand are included to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these sequences in combination with the detailed descriptionpresented herein.

SEQ ID NOs: 1-7 and 1097-1099 all refer to sequences from the lineA3244.

SEQ ID NO: 1 is sequence ID 515O02_region_G2 from line A3244, and isadjacent to the contig containing rhg1.

SEQ ID NO: 2 is sequence ID 240O17_region_G3 from line A3244, andcontains the rhg1, v.1 four exon gene at coding coordinates 45163-45314,45450-45509, 46941-48763, 48975-49573. The amino acid translation forSEQ ID NO: 2 is SEQ ID NO: 1097.

SEQ ID NO: 3 is sequence ID 240O17_region_G3 from line A3244, andcontains the rhg1, v.2 two exon gene at coding coordinates 46798-48763and 48975-49573. The amino acid translation for SEQ ID NO: 3 is SEQ IDNO: 1098.

SEQ ID NO: 4 is sequence ID 318O13_region_A3 from line A3244, containsthe Rhg4 gene at coding coordinates 111805-113968 and 114684-115204, andhas an amino acid translation of SEQ ID NO: 1099.

SEQ ID NO: 5 is sequence ID 240O17_region_G3_(—)8_mRNA, and comprisesthe two rhg1, v.2 exons from the coding sequence portion of SEQ ID NO:3.

SEQ ID NO: 6 is sequence ID 240O17_region_G3_(—)8_cds, and comprises thefour rhg1, v.1 exons from the coding sequence portion of SEQ ID NO: 2.

SEQ ID NO: 7 is sequence ID 318O13_region_A3_(—)17_cds, and comprisesthe Rhg4 coding sequence portion from SEQ ID NO: 4.

SEQ ID NOs: 8-43 and 1100-1115 all refer to rhg1 sequences.

SEQ ID NO: 8 is sequence ID rhg1_A3244_amplicon from line A3244,contains four rhg1, v.1 exons at coding coordinates 113-264, 400-459,1891-3713, and 3925-4523, and has an amino acid translation of SEQ IDNO: 1100 and 1097.

SEQ ID NO: 9 is sequence ID rhg1_A3244_amplicon, contains two rhg1, v.2exons at coding coordinates 1748-3713 and 3925-4523 and has an aminoacid translation of SEQ ID NO: 1101 and 1098.

SEQ ID NO: 10 is sequence ID rhg1_peking_amplicon from the line peking,contains four rhg1, v.1 exons at coding coordinates 113-264, 400-459,1888-3710, and 3903-4501, and has an amino acid translation of SEQ IDNO: 1102.

SEQ ID NO: 11 is sequence ID rhg1_peking_amplicon, contains two rhg1,v.2 exons at coding coordinates 1745-3710 and 3903-4501, and has anamino acid translation of SEQ ID NO: 1103.

SEQ ID NO: 12 is sequence ID rhg1_toyosuzu_amplicon from the linetoyosuzu, contains four rhg1, v.1 exons at coding coordinates 113-264,400-459, 1890-3712, and 3924-4522, and has an amino acid translation ofSEQ ID NO: 1104.

SEQ ID NO: 13 is sequence ID rhg1_toyosuzu_amplicon, contains two rhg1,v.2 exons at coding coordinates 1747-3712 and 3924-4522, and has anamino acid translation of SEQ ID NO: 1105.

SEQ ID NO: 14 is sequence ID rhg1_will_amplicon from the line will,contains four rhg1, v.1 exons at coding coordinates 113-264, 400-459,1891-3713, and 3925-4523, and has an amino acid translation of SEQ IDNO: 1106.

SEQ ID NO: 15 is sequence ID rhg1_will_amplicon, contains two rhg1, v.2exons at coding coordinates 1748-3713 and 3925-4523, and has an aminoacid translation of SEQ ID NO: 1107.

SEQ ID NO: 16 is sequence ID rhg1_a2704_amplicon from the line A2704,contains four rhg1, v.1 exons at coding coordinates 113-264, 400-459,1891-3713, and 3925-4523, and has an amino acid translation of SEQ IDNO: 1108.

SEQ ID NO: 17 is sequence ID rhg1_a2704_amplicon, contains two rhg1, v.2exons at coding coordinates 1748-3713 and 3925-4523, and has an aminoacid translation of SEQ ID NO: 1109.

SEQ ID NO: 18 is sequence ID rhg1_noir_amplicon from the line noir,contains four rhg1, v.1 exons at coding coordinates 113-264, 400-459,1876-3698, and 3910-4508, and has an amino acid translation of SEQ IDNO: 1110.

SEQ ID NO: 19 is sequence ID rhg1_noir_amplicon, contains two rhg1, v.2exons at coding coordinates 1733-3698 and 3910-4508, and has an aminoacid translation of SEQ ID NO: 1111.

SEQ ID NO: 20 is sequence ID rhg1_lee_amplicon from the line lee,contains four rhg1, v.1 exons at coding coordinates 113-264, 400-459,1876-3698, and 3910-4508, and has an amino acid translation of SEQ IDNO: 1112.

SEQ ID NO: 21 is sequence ID rhg1_lee_amplicon, contains two rhg1, v.2exons at coding coordinates 1733-3698 and 3910-4508, and has an aminoacid translation of SEQ ID NO: 1113.

SEQ ID NO: 22 is sequence ID rhg1_pi200499_amplicon from the linePI200499, contains four rhg1, v.1 exons at coding coordinates 113-264,400-459, 1876-3698, and 3910-4508, and has an amino acid translation ofSEQ ID NO: 1114.

SEQ ID NO: 23 is sequence ID rhg1_pi200499_amplicon, contains two rhg1,v.2 exons at coding coordinates 1733-3698 and 3910-4508, and has anamino acid translation of SEQ ID NO: 1115.

SEQ ID NO: 24 is sequence ID 240O17_region_G3_forward_(—)1, is a primerthat hybridizes to coordinates 45051-45077 on contig 240017_region_G3before the start codon, and can be used with SEQ ID NO: 25.

SEQ ID NO: 25 is sequence ID 240O17_region_G3_reverse_(—)1, is a primerthat hybridizes to coordinates 47942-47918 on contig 240017_region_G3,and can be used with SEQ ID NO: 24.

SEQ ID NO: 26 is sequence ID 240O17_region_G3_forward_(—)2, is a primerthat hybridizes to coordinates 47808-47831 on contig 240017_region_G3,and can be used with SEQ ID NO: 27.

SEQ ID NO: 27 is sequence ID 240O17_region_G3_reverse_(—)2, is a primerthat hybridizes to coordinates 49553-49531 of contig 240017_region_G3prior to the stop codon, and can be used with SEQ ID NO: 26.

Primers given by SEQ ID NOs: 24-27 are used to create the amplicons ofSEQ ID NOs: 8-23. The final 22 bases are added to the actual ampliconsin order to simulate the rest of the gene to the stop codon, in order toallow complete translation.

SEQ ID NO: 28 is sequence ID rhg1_A3244_amplicon_cds, which is thecoding sequence portion of SEQ ID NO: 8.

SEQ ID NO: 29 is sequence ID rhg1_peking_amplicon_cds, which is thecoding sequence portion of SEQ ID NO: 10.

SEQ ID NO: 30 is sequence ID rhg1_toyosuzu_amplicon_cds, which is thecoding sequence portion of SEQ ID NO: 12.

SEQ ID NO: 31 is sequence ID rhg1_will_amplicon_cds, which is the codingsequence portion of SEQ ID NO: 14.

SEQ ID NO: 32 is sequence ID rhg1_a2704_amplicon_cds, which is thecoding sequence portion of SEQ ID NO: 16.

SEQ ID NO: 33 is sequence ID rhg1_noir_amplicon_cds, which is the codingsequence portion of SEQ ID NO: 18.

SEQ ID NO: 34 is sequence ID rhg1_lee_amplicon_cds, which is the codingsequence portion of SEQ ID NO: 20.

SEQ ID NO: 35 is sequence ID rhg1_pi200499_amplicon_cds, which is thecoding sequence portion of SEQ ID NO: 22.

SEQ ID NO: 36 is sequence ID rhg1_A3244_amplicon_cds_(—)2, which is thecoding sequence portion of SEQ ID NO: 9.

SEQ ID NO: 37 is sequence ID rhg1_peking_amplicon_cds_(—)2, which is thecoding sequence portion of SEQ ID NO: 11.

SEQ ID NO: 38 is sequence ID rhg1_toyosuzu_amplicon_cds_(—)2, which isthe coding sequence portion of SEQ ID NO: 13.

SEQ ID NO: 39 is sequence ID rhg1_will_amplicon_cds_(—)2, which is thecoding sequence portion of SEQ ID NO: 15.

SEQ ID NO: 40 is sequence ID rhg1_a2704_amplicon_cds_(—)2, which is thecoding sequence portion of SEQ ID NO: 17.

SEQ ID NO: 41 is sequence ID rhg1_noir_amplicon_cds_(—)2, which is thecoding sequence portion of SEQ ID NO: 19.

SEQ ID NO: 42 is sequence ID rhg1_lee_amplicon_cds_(—)2, which is thecoding sequence portion of SEQ ID NO: 21.

SEQ ID NO: 43 is sequence ID rhg1_pi200499_amplicon_cds_(—)2, which isthe coding sequence portion of SEQ ID NO: 23.

SEQ ID NOs: 44-53 and 1116-1119 all refer to Rhg4 sequences.

SEQ ID NO: 44 is sequence ID rhg4_a3244_amplicon from the line A3244,contains Rhg4 at coding coordinates 79-2242 and 2958-3478, is made usingSEQ ID NOs: 48 and 49, and has an amino acid translation of SEQ ID NO:1116 and 1099.

SEQ ID NO: 45 is sequence ID rhg4_Minsoy_amplicon from the line Minsoy,contains Rhg4 at coding coordinates 79-2242 and 2958-3478, is made usingSEQ ID NOs: 48 and 49, and has an amino acid translation of SEQ ID NO:1117.

SEQ ID NO: 46 is sequence ID rhg4_Jack_amplicon from the line Jack,contains Rhg4 at coding coordinates 79-2242 and 2958-3478, is made usingSEQ ID NO: 48 and 49, and has an amino acid translation of SEQ ID NO:1118.

SEQ ID NO: 47 is sequence ID rhg4_peking_amplicon from the line Peking,contains Rhg4 at coding coordinates 79-2242 and 2958-3478, is made usingSEQ ID NOs: 48 and 49, and has an amino acid translation of SEQ ID NO:1119.

SEQ ID NO: 48 is sequence ID 318O13_region_A3_forward, hybridizes tocoordinates 111727-111756 of contig 318O13_region_A3, and is a primerused with SEQ ID NO: 49 to create Rhg4 amplicons.

SEQ ID NO: 49 is sequence ID 318O13_region_A3_reverse, hybridizes tocoordinates 115206-115177 of contig 318O13_region_A3, and is a primerused with SEQ ID NO: 48 to create Rhg4 amplicons.

SEQ ID NO: 50 is sequence ID rhg4_A3244_amplicon_cds, which is thecoding sequence portion of SEQ ID NO: 44.

SEQ ID NO: 51 is sequence ID rhg4_Minsoy_amplicon_cds, which is thecoding sequence portion of SEQ ID NO: 45.

SEQ ID NO: 52 is sequence ID rhg4_Jack_amplicon_cds, which is the codingsequence portion of SEQ ID NO: 46.

SEQ ID NO: 53 is sequence ID rhg4_peking_amplicon_cds, which is thecoding sequence portion of SEQ ID NO: 47.

SEQ ID NO: 1120 is sequence ID consensusLRR, which is a consensussequence for the LRR repeats shown in FIGS. 1 and 2.

SEQ ID NO: 1121 is sequence ID rhg1LRR, which is the amino acid sequenceof the LRR domain shown in FIG. 1.

SEQ ID NO: 1122 is sequence ID Rhg4LRR, which is the amino acid sequenceof the LRR domain shown in FIG. 2.

SEQ ID NO: 1123 is sequence ID 240O17 region_G3_forward_(—)1_b, which isan alternate primer that hybridizes to coordinates 45046-45072 on contig240017_region_G3 before the start codon, and which can be used with SEQID NO: 25.

Table 1 below provides further information on the sequences describedherein.

In table 1, for all rows, “Seq Num” refers to the corresponding SEQ IDNO in the sequence listing.

For rows with SEQ ID NOs: 1-53 and 1120-1123 “Seq ID” refers to the nameof the SEQ ID NO given in the “Seq Num” column.

For rows with SEQ ID NOs: 2-4, 8-23, and 44-47“Coding Sequence” refersto the coordinates of the coding portion of the SEQ ID NO given in the“Seq Num” column, and “AA” refers to the SEQ ID NO that is the aminoacid translation of the SEQ ID NO given in the “Seq Num” column.

For rows with SEQ ID NOs: 24-27 and 1123, “Primer location on240017_region_G3” refers to the coordinates of the 240017_region_G3contig to which the SEQ ID NO given in the “Seq Num” column hybridizes.

For rows with SEQ ID NOs: 48 and 49, “Primer location on318O13_region_A3” refers to the coordinates of the 318013_region_A3contig to which the SEQ ID NO given in the “Seq Num” column hybridizes.

For rows with SEQ ID NOs: 54-400, “Seq ID” refers to the names ofamplicon sequences. Within the Seq ID is the “_” (double lengthunderscore) symbol. The name before this symbol refers to the name ofthe contig in which the amplicon is found, and the numbers after thissymbol refer to the nucleotide location of the SSR on the contig.

For rows with SEQ ID NOs: 401-1096, “Seq ID” refers to the names ofprimer sequences used in PCR to generate the amplicon sequences intable 1. For these rows, the “Seq ID” name contains the same name as theamplicon that is generated by the pair of primers of which the SEQ ID NOreferred to in the first column is a member. The “Seq ID” name alsocontains either “Forward” or “Reverse,” which indicates the orientationof the primer. For these sequences, “location of primer on contig start”and “location of primer on contig end” refer, respectively, to the firstand last base number of the contig on which the primer aligns. TABLE 1Seq Num Seq ID 1 515O02_region_G2 Coding Sequence AA No. 2240O17_region_G3 45163-45314, 45450-45509, 46941-48763, 1097 48975-495733 240O17_region_G3 46798-48763, 48975-49573 1098 4 318O13_region_A3111805-113968, 114684-115204 1099 5 240O17_region_G3_8_mRNA 6240O17_region_G3_8_cds 7 318O13_region_A3_17_cds Coding Sequence AA No.8 rhg1_A3244_amplicon 113-264, 400-459, 1891-3713, 3925-4523 1100 9rhg1_A3244_amplicon 1748-3713, 3925-4523 1101 10 rhg1_peking_amplicon113-264, 400-459, 1888-3710, 3903-4501 1102 11 rhg1_peking_amplicon1745-3710, 3903-4501 1103 12 rhg1_toyosuzu_amplicon 113-264, 400-459,1890-3712, 3924-4522 1104 13 rhg1_toyosuzu_amplicon 1747-3712, 3924-45221105 14 rhg1_will_amplicon 113-264, 400-459, 1891-3713, 3925-4523 110615 rhg1_will_amplicon 1748-3713, 3925-4523 1107 16 rhg1_a2704_amplicon113-264, 400-459, 1891-3713, 3925-4523 1108 17 rhg1_a2704_amplicon1748-3713, 3925-4523 1109 18 rhg1_noir_amplicon 113-264, 400-459,1876-3698, 3910-4508 1110 19 rhg1_noir_amplicon 1733-3698, 3910-45081111 20 rhg1_lee_amplicon 113-264, 400-459, 1876-3698, 3910-4508 1112 21rhg1_lee_amplicon 1733-3698, 3910-4508 1113 22 rhg1_pi200499_amplicon113-264, 400-459, 1876-3698, 3910-4508 1114 23 rhg1_pi200499_amplicon1733-3698, 3910-4508 1115 Primer location on 240O17_region_G3 24240O17_region_G3_forward_1 45051-45077 25 240O17_region_G3_reverse_147942-47918 26 240O17_region_G3_forward_2 47808-47831 27240O17_region_G3_reverse_2 49553-49531 28 rhg1_A3244_amplicon_cds 29rhg1_peking_amplicon_cds 30 rhg1_toyosuzu_amplicon_cds 31rhg1_will_amplicon_cds 32 rhg1_a2704_amplicon_cds 33rhg1_noir_amplicon_cds 34 rhg1_lee_amplicon_cds 35rhg1_pi200499_amplicon_cds 36 rhg1_A3244_amplicon_cds_2 37rhg1_peking_amplicon_cds_2 38 rhg1_toyosuzu_amplicon_cds_2 39rhg1_will_amplicon_cds_2 40 rhg1_a2704_amplicon_cds_2 41rhg1_noir_amplicon_cds_2 42 rhg1_lee_amplicon_cds_2 43rhg1_pi200499_amplicon_cds_2 Coding Sequence AA No. 44rhg4_a3244_amplicon 79-2242, 2958-3478 1116 45 rhg4_Minsoy_amplicon79-2242, 2958-3478 1117 46 rhg4_Jack_amplicon 79-2242, 2958-3478 1118 47rhg4_peking_amplicon 79-2242, 2958-3478 1119 Primer location on318O13_region_A3 48 318O13_region_A3_forward 111727-111756 49318O13_region_A3_reverse 115206-115177 50 rhg4_A3244_amplicon_cds 51rhg4_Minsoy_amplicon_cds 52 rhg4_Jack_amplicon_cds 53rhg4_peking_amplicon_cds 54 240O17_region_G3_289711_11 55240O17_region_G3_236585_14 56 240O17_region_G3_168772_13 57240O17_region_G3_332420_21 58 240O17_region_G3_228126_18 59240O17_region_G3_139723_11 60 240O17_region_G3_280585_14 61240O17_region_G3_70509_14 62 240O17_region_G3_50537_17 63240O17_region_G3_231556_17 64 240O17_region_G3_117057_11 65240O17_region_G3_23092_13 66 240O17_region_G3_297741_14 67240O17_region_G3_206502_14 68 240O17_region_G3_221223_13 69240O17_region_G3_169084_14 70 240O17_region_G3_94891_14 71240O17_region_G3_281852_61 72 240O17_region_G3_46583_12 73240O17_region_G3_306835_13 74 240O17_region_G3_85471_12 75240O17_region_G3_257208_12 76 240O17_region_G3_150390_17 77240O17_region_G3_34697_75 78 240O17_region_G3_150374_13 79240O17_region_G3_40513_22 80 240O17_region_G3_268602_14 81240O17_region_G3_25357_13 82 240O17_region_G3_137548_13 83240O17_region_G3_139131_13 84 240O17_region_G3_203855_12 85240O17_region_G3_199049_15 86 240O17_region_G3_320907_12 87240O17_region_G3_16407_17 88 240O17_region_G3_206516_17 89240O17_region_G3_264495_13 90 240O17_region_G3_156785_13 91240O17_region_G3_187129_12 92 240O17_region_G3_214106_13 93240O17_region_G3_149013_12 94 240O17_region_G3_326352_16 95240O17_region_G3_278962_12 96 240O17_region_G3_256930_13 97240O17_region_G3_29646_14 98 240O17_region_G3_29618_13 99240O17_region_G3_108561_14 100 240O17_region_G3_143975_14 101240O17_region_G3_108431_20 102 240O17_region_G3_281764_11 103240O17_region_G3_130058_15 104 240O17_region_G3_310590_52 105240O17_region_G3_313405_14 106 240O17_region_G3_302190_13 107240O17_region_G3_225343_17 108 240O17_region_G3_208823_14 109240O17_region_G3_74285_11 110 240O17_region_G3_109052_16 111240O17_region_G3_6395_12 112 240O17_region_G3_244905_16 113240O17_region_G3_244956_13 114 240O17_region_G3_117220_13 115240O17_region_G3_134707_14 116 240O17_region_G3_35078_13 117240O17_region_G3_210506_16 118 240O17_region_G3_116961_26 119240O17_region_G3_51073_13 120 240O17_region_G3_55291_15 121240O17_region_G3_229651_18 122 240O17_region_G3_303308_19 123240O17_region_G3_168373_20 124 240O17_region_G3_253333_17 125240O17_region_G3_5791_13 126 240O17_region_G3_206841_19 127240O17_region_G3_202827_12 128 240O17_region_G3_322656_13 129240O17_region_G3_111841_14 130 240O17_region_G3_192719_13 131240O17_region_G3_195630_17 132 240O17_region_G3_69999_13 133240O17_region_G3_11176_13 134 240O17_region_G3_228643_13 135240O17_region_G3_88478_19 136 240O17_region_G3_108950_13 137240O17_region_G3_121054_14 138 240O17_region_G3_188337_14 139240O17_region_G3_255944_21 140 240O17_region_G3_219518_14 141240O17_region_G3_235601_15 142 240O17_region_G3_301529_13 143240O17_region_G3_94795_14 144 240O17_region_G3_46703_23 145240O17_region_G3_59616_14 146 240O17_region_G3_296933_15 147240O17_region_G3_192428_17 148 240O17_region_G3_191490_14 149240O17_region_G3_201115_11 150 240O17_region_G3_72882_15 151240O17_region_G3_69514_13 152 240O17_region_G3_37699_47 153240O17_region_G3_11301_29 154 240O17_region_G3_141875_12 155240O17_region_G3_98090_18 156 240O17_region_G3_43298_35 157240O17_region_G3_262094_11 158 240O17_region_G3_262079_15 159240O17_region_G3_59090_12 160 240O17_region_G3_245723_13 161240O17_region_G3_194628_54 162 240O17_region_G3_4566_16 163240O17_region_G3_96209_14 164 240O17_region_G3_248715_17 165240O17_region_G3_71410_40 166 240O17_region_G3_226519_13 167240O17_region_G3_11282_19 168 240O17_region_G3_170504_12 169240O17_region_G3_40864_14 170 240O17_region_G3_13529_14 171240O17_region_G3_22858_14 172 240O17_region_G3_309211_13 173240O17_region_G3_55568_26 174 240O17_region_G3_73238_16 175240O17_region_G3_52488_19 176 318O13_region_A3_471518_14 177318O13_region_A3_231599_23 178 318O13_region_A3_375912_13 179318O13_region_A3_180013_12 180 318O13_region_A3_171606_14 181318O13_region_A3_416256_13 182 318O13_region_A3_231395_15 183318O13_region_A3_5502_47 184 318O13_region_A3_93061_14 185318O13_region_A3_111684_19 186 318O13_region_A3_69328_14 187318O13_region_A3_36529_17 188 318O13_region_A3_139128_12 189318O13_region_A3_495674_13 190 318O13_region_A3_187577_13 191318O13_region_A3_453036_14 192 318O13_region_A3_374041_13 193318O13_region_A3_3412_11 194 318O13_region_A3_276495_28 195318O13_region_A3_151839_17 196 318O13_region_A3_292912_12 197318O13_region_A3_104560_12 198 318O13_region_A3_65193_11 199318O13_region_A3_110573_70 200 318O13_region_A3_65117_12 201318O13_region_A3_490837_16 202 318O13_region_A3_107448_11 203318O13_region_A3_331_23 204 318O13_region_A3_193470_13 205318O13_region_A3_183305_14 206 318O13_region_A3_55050_14 207318O13_region_A3_224693_21 208 318O13_region_A3_207216_12 209318O13_region_A3_4654_22 210 318O13_region_A3_408959_13 211318O13_region_A3_132288_22 212 318O13_region_A3_292822_20 213318O13_region_A3_311076_12 214 318O13_region_A3_509623_13 215318O13_region_A3_190404_14 216 318O13_region_A3_164916_15 217318O13_region_A3_21028_13 218 318O13_region_A3_208012_17 219318O13_region_A3_484089_14 220 318O13_region_A3_332780_17 221318O13_region_A3_480137_37 222 318O13_region_A3_441056_14 223318O13_region_A3_77486_11 224 318O13_region_A3_272468_11 225318O13_region_A3_425319_17 226 318O13_region_A3_413879_31 227318O13_region_A3_80477_64 228 318O13_region_A3_277272_50 229318O13_region_A3_509642_13 230 318O13_region_A3_321771_14 231318O13_region_A3_26788_12 232 318O13_region_A3_262706_16 233318O13_region_A3_243928_16 234 318O13_region_A3_23246_14 235318O13_region_A3_165406_12 236 318O13_region_A3_486294_14 237318O13_region_A3_46754_12 238 318O13_region_A3_381116_15 239318O13_region_A3_350369_11 240 318O13_region_A3_138841_13 241318O13_region_A3_12158_14 242 318O13_region_A3_315368_13 243318O13_region_A3_307549_13 244 318O13_region_A3_159857_14 245318O13_region_A3_140551_15 246 318O13_region_A3_279869_11 247318O13_region_A3_78292_35 248 318O13_region_A3_185019_12 249318O13_region_A3_409164_13 250 318O13_region_A3_75392_14 251318O13_region_A3_231320_12 252 318O13_region_A3_381102_14 253318O13_region_A3_491826_15 254 318O13_region_A3_56365_21 255318O13_region_A3_372628_15 256 318O13_region_A3_302609_11 257318O13_region_A3_341804_11 258 318O13_region_A3_217037_11 259318O13_region_A3_264929_68 260 318O13_region_A3_55499_12 261318O13_region_A3_295634_14 262 318O13_region_A3_269358_15 263318O13_region_A3_457009_24 264 318O13_region_A3_176598_14 265318O13_region_A3_278266_12 266 318O13_region_A3_391810_12 267318O13_region_A3_269485_15 268 318O13_region_A3_359247_17 269318O13_region_A3_315094_13 270 318O13_region_A3_307823_13 271318O13_region_A3_248588_15 272 318O13_region_A3_252426_85 273318O13_region_A3_513314_16 274 318O13_region_A3_68183_14 275318O13_region_A3_471191_13 276 318O13_region_A3_163547_18 277318O13_region_A3_417867_15 278 318O13_region_A3_332465_14 279318O13_region_A3_207697_14 280 318O13_region_A3_277229_43 281318O13_region_A3_36366_11 282 318O13_region_A3_91970_12 283318O13_region_A3_211533_11 284 318O13_region_A3_336301_11 285318O13_region_A3_441603_14 286 318O13_region_A3_468354_15 287318O13_region_A3_188983_18 288 318O13_region_A3_115502_17 289318O13_region_A3_163006_13 290 318O13_region_A3_119283_14 291318O13_region_A3_491126_11 292 318O13_region_A3_99512_21 293318O13_region_A3_280291_17 294 318O13_region_A3_138443_19 295318O13_region_A3_115973_14 296 318O13_region_A3_329977_14 297318O13_region_A3_205203_14 298 318O13_region_A3_153114_12 299318O13_region_A3_34581_13 300 318O13_region_A3_292577_19 301318O13_region_A3_445391_20 302 318O13_region_A3_350540_17 303318O13_region_A3_453879_15 304 318O13_region_A3_201246_13 305318O13_region_A3_326020_13 306 318O13_region_A3_503801_14 307318O13_region_A3_302400_52 308 318O13_region_A3_448857_15 309318O13_region_A3_48364_14 310 318O13_region_A3_251804_48 311318O13_region_A3_382583_13 312 318O13_region_A3_124737_14 313318O13_region_A3_124766_13 314 318O13_region_A3_461351_16 315318O13_region_A3_64953_19 316 318O13_region_A3_366586_13 317318O13_region_A3_46190_15 318 318O13_region_A3_81016_11 319318O13_region_A3_134426_14 320 318O13_region_A3_292724_14 321318O13_region_A3_187096_17 322 318O13_region_A3_381693_13 323318O13_region_A3_361286_33 324 318O13_region_A3_482668_14 325318O13_region_A3_128002_12 326 318O13_region_A3_499270_14 327318O13_region_A3_231650_12 328 318O13_region_A3_199851_13 329318O13_region_A3_324629_13 330 318O13_region_A3_374190_19 331318O13_region_A3_460603_13 332 318O13_region_A3_108681_14 333318O13_region_A3_459791_47 334 318O13_region_A3_4257_20 335318O13_region_A3_238810_14 336 318O13_region_A3_245817_14 337318O13_region_A3_245956_14 338 318O13_region_A3_74148_14 339318O13_region_A3_74089_15 340 318O13_region_A3_241686_12 341318O13_region_A3_47476_12 342 318O13_region_A3_164550_12 343318O13_region_A3_101255_15 344 515O02_region_G2_16189_11 345515O02_region_G2_71925_13 346 515O02_region_G2_4707_12 347515O02_region_G2_118904_18 348 515O02_region_G2_13655_17 349515O02_region_G2_53900_13 350 515O02_region_G2_8079_14 351515O02_region_G2_9969_28 352 515O02_region_G2_72308_77 353515O02_region_G2_99475_19 354 515O02_region_G2_118615_18 355515O02_region_G2_119001_46 356 515O02_region_G2_118958_43 357515O02_region_G2_17197_13 358 515O02_region_G2_105163_29 359515O02_region_G2_111335_13 360 515O02_region_G2_106396_13 361515O02_region_G2_59229_17 362 515O02_region_G2_73795_20 363515O02_region_G2_85664_20 364 515O02_region_G2_36921_17 365515O02_region_G2_124150_19 366 515O02_region_G2_5089_14 367515O02_region_G2_58221_15 368 515O02_region_G2_96139_14 369515O02_region_G2_70595_13 370 515O02_region_G2_4340_15 371515O02_region_G2_90417_11 372 515O02_region_G2_49711_17 373515O02_region_G2_63053_13 374 515O02_region_G2_63076_14 375515O02_region_G2_44442_12 376 515O02_region_G2_44422_19 377515O02_region_G2_44158_19 378 515O02_region_G2_44141_17 379515O02_region_G2_90762_17 380 515O02_region_G2_106241_14 381515O02_region_G2_109676_12 382 515O02_region_G2_86242_14 383515O02_region_G2_83109_12 384 515O02_region_G2_10461_15 385515O02_region_G2_67608_15 386 515O02_region_G2_63275_46 387515O02_region_G2_62405_14 388 515O02_region_G2_33563_12 389515O02_region_G2_33146_14 390 515O02_region_G2_102179_29 391515O02_region_G2_2646_15 392 515O02_region_G2_76652_24 393515O02_region_G2_66280_14 394 515O02_region_G2_54768_13 395515O02_region_G2_62580_14 396 515O02_region_G2_34598_55 397515O02_region_G2_77680_13 398 515O02_region_G2_77693_12 399515O02_region_G2_97392_14 400 515O02_region_G2_97359_15 location ofprimer location of primer on contig start on contig end 401240O17_region_G3_289711_11_Forward_Primer 289637 289661 402240O17_region_G3_289711_11_Reverse_Primer 289756 289732 403240O17_region_G3_236585_14_Forward_Primer 236511 236535 404240O17_region_G3_236585_14_Reverse_Primer 236638 236614 405240O17_region_G3_168772_13_Forward_Primer 168683 168707 406240O17_region_G3_168772_13_Reverse_Primer 168811 168786 407240O17_region_G3_332420_21_Forward_Primer 332375 332399 408240O17_region_G3_332420_21_Reverse_Primer 332505 332481 409240O17_region_G3_228126_18_Forward_Primer 228048 228072 410240O17_region_G3_228126_18_Reverse_Primer 228182 228158 411240O17_region_G3_139723_11_Forward_Primer 139666 139690 412240O17_region_G3_139723_11_Reverse_Primer 139802 139778 413240O17_region_G3_280585_14_Forward_Primer 280524 280550 414240O17_region_G3_280585_14_Reverse_Primer 280661 280637 415240O17_region_G3_70509_14_Forward_Primer 70478 70502 416240O17_region_G3_70509_14_Reverse_Primer 70616 70592 417240O17_region_G3_50537_17_Forward_Primer 50455 50479 418240O17_region_G3_50537_17_Reverse_Primer 50593 50569 419240O17_region_G3_231556_17_Forward_Primer 231468 231492 420240O17_region_G3_231556_17_Reverse_Primer 231606 231582 421240O17_region_G3_117057_11_Forward_Primer 117029 117053 422240O17_region_G3_117057_11_Reverse_Primer 117169 117145 423240O17_region_G3_23092_13_Forward_Primer 23010 23034 424240O17_region_G3_23092_13_Reverse_Primer 23151 23127 425240O17_region_G3_297741_14_Forward_Primer 297680 297704 426240O17_region_G3_297741_14_Reverse_Primer 297823 297799 427240O17_region_G3_206502_14_Forward_Primer 206456 206480 428240O17_region_G3_206502_14_Reverse_Primer 206600 206581 429240O17_region_G3_221223_13_Forward_Primer 221134 221158 430240O17_region_G3_221223_13_Reverse_Primer 221278 221254 431240O17_region_G3_169084_14_Forward_Primer 169051 169075 432240O17_region_G3_169084_14_Reverse_Primer 169196 169173 433240O17_region_G3_94891_14_Forward_Primer 94784 94808 434240O17_region_G3_94891_14_Reverse_Primer 94929 94905 435240O17_region_G3_7439_12_Forward_Primer 7397 7421 436240O17_region_G3_7439_12_Reverse_Primer 7542 7518 437240O17_region_G3_281852_61_Forward_Primer 281797 281821 438240O17_region_G3_281852_61_Reverse_Primer 281943 281919 439240O17_region_G3_46583_12_Forward_Primer 46554 46578 440240O17_region_G3_46583_12_Reverse_Primer 46700 46676 441240O17_region_G3_306835_13_Forward_Primer 306727 306751 442240O17_region_G3_306835_13_Reverse_Primer 306874 306849 443240O17_region_G3_85471_12_Forward_Primer 85359 85383 444240O17_region_G3_85471_12_Reverse_Primer 85507 85483 445240O17_region_G3_257208_12_Forward_Primer 257129 257153 446240O17_region_G3_257208_12_Reverse_Primer 257278 257254 447240O17_region_G3_150390_17_Forward_Primer 150327 150351 448240O17_region_G3_150390_17_Reverse_Primer 150476 150452 449240O17_region_G3_34697_75_Forward_Primer 34662 34685 450240O17_region_G3_34697_75_Reverse_Primer 34811 34787 451240O17_region_G3_150374_13_Forward_Primer 150327 150351 452240O17_region_G3_150374_13_Reverse_Primer 150476 150452 453240O17_region_G3_40513_22_Forward_Primer 40422 40446 454240O17_region_G3_40513_22_Reverse_Primer 40572 40548 455240O17_region_G3_268602_14_Forward_Primer 268555 268579 456240O17_region_G3_268602_14_Reverse_Primer 268705 268681 457240O17_region_G3_25357_13_Forward_Primer 25271 25295 458240O17_region_G3_25357_13_Reverse_Primer 25422 25402 459240O17_region_G3_137548_13_Forward_Primer 139088 139111 459240O17_region_G3_137548_13_Forward_Primer 137505 137528 460240O17_region_G3_137548_13_Reverse_Primer 139239 139215 460240O17_region_G3_137548_13_Reverse_Primer 137656 137632 461240O17_region_G3_139131_13_Forward_Primer 139088 139111 462240O17_region_G3_139131_13_Reverse_Primer 139239 139215 463240O17_region_G3_203855_12_Forward_Primer 203749 203773 464240O17_region_G3_203855_12_Reverse_Primer 203901 203877 465240O17_region_G3_199049_15_Forward_Primer 199008 199033 466240O17_region_G3_199049_15_Reverse_Primer 199160 199136 467240O17_region_G3_320907_12_Forward_Primer 320885 320906 468240O17_region_G3_320907_12_Reverse_Primer 321038 321015 469240O17_region_G3_16407_17_Forward_Primer 16330 16354 470240O17_region_G3_16407_17_Reverse_Primer 16483 16459 471240O17_region_G3_206516_17_Forward_Primer 206482 206506 472240O17_region_G3_206516_17_Reverse_Primer 206635 206616 473240O17_region_G3_264495_13_Forward_Primer 264423 264447 474240O17_region_G3_264495_13_Reverse_Primer 264577 264553 475240O17_region_G3_156785_13_Forward_Primer 156713 156737 476240O17_region_G3_156785_13_Reverse_Primer 156868 156844 477240O17_region_G3_187129_12_Forward_Primer 187068 187092 478240O17_region_G3_187129_12_Reverse_Primer 187223 187199 479240O17_region_G3_214106_13_Forward_Primer 214042 214067 480240O17_region_G3_214106_13_Reverse_Primer 214197 214173 481240O17_region_G3_149013_12_Forward_Primer 148898 148922 482240O17_region_G3_149013_12_Reverse_Primer 149053 149027 483240O17_region_G3_326352_16_Forward_Primer 326311 326335 484240O17_region_G3_326352_16_Reverse_Primer 326467 326443 485240O17_region_G3_278962_12_Forward_Primer 278933 278957 486240O17_region_G3_278962_12_Reverse_Primer 279089 279065 487240O17_region_G3_256930_13_Forward_Primer 256850 256874 488240O17_region_G3_256930_13_Reverse_Primer 257006 256982 489240O17_region_G3_29646_14_Forward_Primer 29589 29613 490240O17_region_G3_29646_14_Reverse_Primer 29746 29721 491240O17_region_G3_29618_13_Forward_Primer 29589 29613 49240O17_region_G3_29618_13_Reverse_Primer 29746 29721 493240O17_region_G3_108561_14_Forward_Primer 108518 108542 494240O17_region_G3_108561_14_Reverse_Primer 108675 108651 495240O17_region_G3_143975_14_Forward_Primer 143939 143964 496240O17_region_G3_143975_14_Reverse_Primer 144096 144072 497240O17_region_G3_108431_20_Forward_Primer 108362 108386 498240O17_region_G3_108431_20_Reverse_Primer 108520 108497 499240O17_region_G3_281764_11_Forward_Primer 281645 281669 500240O17_region_G3_281764_11_Reverse_Primer 281803 281779 501240O17_region_G3_130058_15_Forward_Primer 129994 130018 502240O17_region_G3_130058_15_Reverse_Primer 130153 130129 503240O17_region_G3_310590_52_Forward_Primer 310533 310557 504240O17_region_G3_310590_52_Reverse_Primer 310692 310668 505240O17_region_G3_313405_14_Forward_Primer 313345 313369 506240O17_region_G3_313405_14_Reverse_Primer 313505 313481 507240O17_region_G3_302190_13_Forward_Primer 302093 302119 508240O17_region_G3_302190_13_Reverse_Primer 302253 302229 509240O17_region_G3_225343_17_Forward_Primer 225315 225338 510240O17_region_G3_225343_17_Reverse_Primer 225475 225451 511240O17_region_G3_208823_14_Forward_Primer 208760 208784 512240O17_region_G3_208823_14_Reverse_Primer 208921 208897 513240O17_region_G3_74285_11_Forward_Primer 74220 74244 514240O17_region_G3_74285_11_Reverse_Primer 74382 74358 515240O17_region_G3_109052_16_Forward_Primer 108999 109023 516240O17_region_G3_109052_16_Reverse_Primer 109161 109137 517240O17_region_G3_6395_12_Forward_Primer 6285 6309 518240O17_region_G3_6395_12_Reverse_Primer 6447 6423 519240O17_region_G3_244905_16_Forward_Primer 244865 244890 520240O17_region_G3_244905_16_Reverse_Primer 245028 245004 521240O17_region_G3_244956_13_Forward_Primer 244865 244890 522240O17_region_G3_244956_13_Reverse_Primer 245028 245004 523240O17_region_G3_117220_13_Forward_Primer 117175 117199 524240O17_region_G3_117220_13_Reverse_Primer 117339 117315 525240O17_region_G3_134707_14_Forward_Primer 134584 134608 526240O17_region_G3_134707_14_Reverse_Primer 134749 134725 527240O17_region_G3_35078_13_Forward_Primer 34990 35013 528240O17_region_G3_35078_13_Reverse_Primer 35157 35133 529240O17_region_G3_210506_16_Forward_Primer 210477 210501 530240O17_region_G3_210506_16_Reverse_Primer 210644 210620 531240O17_region_G3_116961_26_Forward_Primer 116885 116909 532240O17_region_G3_116961_26_Reverse_Primer 117053 117029 533240O17_region_G3_51073_13_Forward_Primer 50979 51003 534240O17_region_G3_51073_13_Reverse_Primer 51147 51123 535240O17_region_G3_55291_15_Forward_Primer 55164 55188 536240O17_region_G3_55291_15_Reverse_Primer 55333 55309 537240O17_region_G3_229651_18_Forward_Primer 229615 229639 538240O17_region_G3_229651_18_Reverse_Primer 229784 229760 539240O17_region_G3_303308_19_Forward_Primer 303284 303307 540240O17_region_G3_303308_19_Reverse_Primer 303454 303429 541240O17_region_G3_168373_20_Forward_Primer 168262 168286 542240O17_region_G3_168373_20_Reverse_Primer 168432 168408 543240O17_region_G3_253333_17_Forward_Primer 253257 253281 544240O17_region_G3_253333_17_Reverse_Primer 253428 253404 545240O17_region_G3_5791_13_Forward_Primer 5766 5790 546240O17_region_G3_5791_13_Reverse_Primer 5937 5912 547240O17_region_G3_206841_19_Forward_Primer 206821 206840 548240O17_region_G3_206841_19_Reverse_Primer 206993 206969 549240O17_region_G3_202827_12_Forward_Primer 202782 202806 550240O17_region_G3_202827_12_Reverse_Primer 202956 202932 551240O17_region_G3_322656_13_Forward_Primer 322572 322598 552240O17_region_G3_322656_13_Reverse_Primer 322748 322724 553240O17_region_G3_111841_14_Forward_Primer 111709 111733 554240O17_region_G3_111841_14_Reverse_Primer 111886 111861 555240O17_region_G3_192719_13_Forward_Primer 192589 192613 556240O17_region_G3_192719_13_Reverse_Primer 192767 192743 557240O17_region_G3_195630_17_Forward_Primer 195490 195514 558240O17_region_G3_195630_17_Reverse_Primer 195672 195648 559240O17_region_G3_69999_13_Forward_Primer 69858 69881 560240O17_region_G3_69999_13_Reverse_Primer 70040 70016 561240O17_region_G3_11176_13_Forward_Primer 11060 11084 562240O17_region_G3_11176_13_Reverse_Primer 11243 11219 563240O17_region_G3_228643_13_Forward_Primer 228529 228553 564240O17_region_G3_228643_13_Reverse_Primer 228713 228689 565240O17_region_G3_88478_19_Forward_Primer 88378 88402 566240O17_region_G3_88478_19_Reverse_Primer 88562 88538 567240O17_region_G3_108950_13_Forward_Primer 108838 108858 568240O17_region_G3_108950_13_Reverse_Primer 109023 108998 569240O17_region_G3_121054_14_Forward_Primer 120911 120935 570240O17_region_G3_121054_14_Reverse_Primer 121096 121072 571240O17_region_G3_188337_14_Forward_Primer 188204 188228 572240O17_region_G3_188337_14_Reverse_Primer 191544 191520 572240O17_region_G3_188337_14_Reverse_Primer 188391 188367 573240O17_region_G3_255944_21_Forward_Primer 255879 255903 574240O17_region_G3_255944_21_Reverse_Primer 256068 256044 575240O17_region_G3_219518_14_Forward_Primer 219420 219444 576240O17_region_G3_219518_14_Reverse_Primer 219609 219585 577240O17_region_G3_235601_15_Forward_Primer 235483 235507 578240O17_region_G3_235601_15_Reverse_Primer 235673 235649 579240O17_region_G3_301529_13_Forward_Primer 301498 301522 580240O17_region_G3_301529_13_Reverse_Primer 301689 301665 581240O17_region_G3_94795_14_Forward_Primer 94735 94756 582240O17_region_G3_94795_14_Reverse_Primer 94929 94905 583240O17_region_G3_46703_23_Forward_Primer 46676 46700 584240O17_region_G3_46703_23_Reverse_Primer 46870 46846 585240O17_region_G3_59616_14_Forward_Primer 59539 59563 586240O17_region_G3_59616_14_Reverse_Primer 59738 59714 587240O17_region_G3_296933_15_Forward_Primer 296908 296932 588240O17_region_G3_296933_15_Reverse_Primer 297113 297089 589240O17_region_G3_192428_17_Forward_Primer 192402 192426 590240O17_region_G3_192428_17_Reverse_Primer 192613 192589 591240O17_region_G3_191490_14_Forward_Primer 191332 191356 592240O17_region_G3_191490_14_Reverse_Primer 191544 191520 593240O17_region_G3_201115_11_Forward_Primer 200994 201018 594240O17_region_G3_201115_11_Reverse_Primer 201214 201189 595240O17_region_G3_72882_15_Forward_Primer 72848 72874 596240O17_region_G3_72882_15_Reverse_Primer 73068 73042 597240O17_region_G3_69514_13_Forward_Primer 69411 69437 598240O17_region_G3_69514_13_Reverse_Primer 69632 69608 599240O17_region_G3_37699_47_Forward_Primer 37601 37625 600240O17_region_G3_37699_47_Reverse_Primer 37827 37802 601240O17_region_G3_11301_29_Forward_Primer 11274 11300 602240O17_region_G3_11301_29_Reverse_Primer 11501 11477 603240O17_region_G3_141875_12_Forward_Primer 141729 141750 604240O17_region_G3_141875_12_Reverse_Primer 141964 141939 605240O17_region_G3_98090_18_Forward_Primer 98037 98062 606240O17_region_G3_98090_18_Reverse_Primer 98274 98250 607240O17_region_G3_43298_35_Forward_Primer 43144 43168 608240O17_region_G3_43298_35_Reverse_Primer 43387 43363 609240O17_region_G3_262094_11_Forward_Primer 261989 262014 610240O17_region_G3_262094_11_Reverse_Primer 262236 262211 611240O17_region_G3_262079_15_Forward_Primer 261989 262014 612240O17_region_G3_262079_15_Reverse_Primer 262236 262211 613240O17_region_G3_59090_12_Forward_Primer 58986 59012 614240O17_region_G3_59090_12_Reverse_Primer 59248 59224 615240O17_region_G3_245723_13_Forward_Primer 245502 245526 616240O17_region_G3_245723_13_Reverse_Primer 245766 245742 617240O17_region_G3_194628_54_Forward_Primer 194581 194607 618240O17_region_G3_194628_54_Reverse_Primer 194846 194822 619240O17_region_G3_4566_16_Forward_Primer 4455 4479 620240O17_region_G3_4566_16_Reverse_Primer 4722 4696 621240O17_region_G3_96209_14_Forward_Primer 96119 96143 622240O17_region_G3_96209_14_Reverse_Primer 96392 96368 623240O17_region_G3_248715_17_Forward_Primer 248633 248657 624240O17_region_G3_248715_17_Reverse_Primer 248906 248882 625240O17_region_G3_71410_40_Forward_Primer 71357 71379 626240O17_region_G3_71410_40_Reverse_Primer 71636 71611 627240O17_region_G3_226519_13_Forward_Primer 226315 226339 628240O17_region_G3_226519_13_Reverse_Primer 226598 226574 629240O17_region_G3_11282_19_Forward_Primer 11217 11242 630240O17_region_G3_11282_19_Reverse_Primer 11501 11477 631240O17_region_G3_170504_12_Forward_Primer 170409 170433 632240O17_region_G3_170504_12_Reverse_Primer 170694 170671 633240O17_region_G3_40864_14_Forward_Primer 40652 40678 634240O17_region_G3_40864_14_Reverse_Primer 40938 40912 635240O17_region_G3_13529_14_Forward_Primer 13332 13356 636240O17_region_G3_13529_14_Reverse_Primer 13622 13598 637240O17_region_G3_22858_14_Forward_Primer 22675 22699 638240O17_region_G3_22858_14_Reverse_Primer 22966 22942 639240O17_region_G3_309211_13_Forward_Primer 309092 309118 640240O17_region_G3_309211_13_Reverse_Primer 309383 309358 641240O17_region_G3_55568_26_Forward_Primer 55375 55399 642240O17_region_G3_55568_26_Reverse_Primer 55667 55642 643240O17_region_G3_73238_16_Forward_Primer 73043 73069 644240O17_region_G3_73238_16_Reverse_Primer 73342 73318 645240O17_region_G3_352488_19_Forward_Primer 52413 52437 646240O17_region_G3_52488_19_Reverse_Primer 52712 52688 647318O13_region_A3_471518_14_Forward_Primer_Seq 471464 471488 648318O13_region_A3_471518_14_Reverse_Primer_Seq 471567 471541 649318O13_region_A3_231599_23_Forward_Primer_Seq 231568 231592 650318O13_region_A3_231599_23_Reverse_Primer_Seq 231672 231651 651318O13_region_A3_375912_13_Forward_Primer_Seq 375845 375865 652318O13_region_A3_375912_13_Reverse_Primer_Seq 375954 375932 653318O13_region_A3_180013_12_Forward_Primer_Seq 179951 179974 654318O13_region_A3_180013_12_Reverse_Primer_Seq 180060 180038 655318O13_region_A3_171606_14_Forward_Primer_Seq 171545 171569 656318O13_region_A3_171606_14_Reverse_Primer_Seq 171657 171633 657318O13_region_A3_416256_13_Forward_Primer_Seq 416180 416203 658318O13_region_A3_416256_13_Reverse_Primer_Seq 416293 416269 659318O13_region_A3_231395_15_Forward_Primer_Seq 231339 231363 660318O13_region_A3_231395_15_Reverse_Primer_Seq 231461 231438 661318O13_region_A3_5502_47_Forward_Primer_Seq 5461 5485 662318O13_region_A3_5502_47_Reverse_Primer_Seq 5585 5561 663318O13_region_A3_93061_14_Forward_Primer_Seq 92988 93012 664318O13_region_A3_93061_14_Reverse_Primer_Seq 93112 93090 665318O13_region_A3_111684_19_Forward_Primer_Seq 111646 111670 666318O13_region_A3_111684_19_Reverse_Primer_Seq 111772 111748 667318O13_region_A3_69328_14_Forward_Primer_Seq 69246 69269 668318O13_region_A3_69328_14_Reverse_Primer_Seq 69373 69349 669318O13_region_A3_36529_17_Forward_Primer_Seq 36488 36512 670318O13_region_A3_36529_17_Reverse_Primer_Seq 36617 36593 671318O13_region_A3_139128_12_Forward_Primer_Seq 139043 139067 672318O13_region_A3_139128_12_Reverse_Primer_Seq 139174 139150 673318O13_region_A3_495674_13_Forward_Primer_Seq 495592 495616 674318O13_region_A3_495674_13_Reverse_Primer_Seq 495723 495699 675318O13_region_A3_187577_13_Forward_Primer_Seq 187482 187506 676318O13_region_A3_187577_13_Reverse_Primer_Seq 187613 187590 677318O13_region_A3_453036_14_Forward_Primer_Seq 452999 453023 678318O13_region_A3_453036_14_Reverse_Primer_Seq 453132 453108 679318O13_region_A3_374041_13_Forward_Primer_Seq 373964 373988 680318O13_region_A3_374041_13_Reverse_Primer_Seq 374097 374073 681318O13_region_A3_3412_11_Forward_Primer_Seq 3319 3341 682318O13_region_A3_3412_11_Reverse_Primer_Seq 3454 3430 683318O13_region_A3_276495_28_Forward_Primer_Seq 276462 276485 684318O13_region_A3_276495_28_Reverse_Primer_Seq 276598 276574 685318O13_region_A3_151839_17_Forward_Primer_Seq 151744 151768 686318O13_region_A3_151839_17_Reverse_Primer_Seq 151882 151858 687318O13_region_A3_292912_12_Forward_Primer_Seq 292875 292899 688318O13_region_A3_292912_12_Reverse_Primer_Seq 293014 292990 689318O13_region_A3_104560_12_Forward_Primer_Seq 104464 104488 690318O13_region_A3_104560_12_Reverse_Primer_Seq 104604 104580 691318O13_region_A3_65193_11_Forward_Primer_Seq 65155 65179 692318O13_region_A3_65193_11_Reverse_Primer_Seq 65295 65271 693318O13_region_A3_110573_70_Forward_Primer_Seq 110533 110559 694318O13_region_A3_110573_70_Reverse_Primer_Seq 110674 110648 695318O13_region_A3_65117_12_Forward_Primer_Seq 65034 65058 696318O13_region_A3_65117_12_Reverse_Primer_Seq 65177 65153 697318O13_region_A3_490837_16_Forward_Primer_Seq 490762 490786 698318O13_region_A3_490837_16_Reverse_Primer_Seq 490905 490881 699318O13_region_A3_107448_11_Forward_Primer_Seq 107385 107411 700318O13_region_A3_107448_11_Reverse_Primer_Seq 107529 107505 701318O13_region_A3_331_23_Forward_Primer_Seq 276 301 702318O13_region_A3_331_23_Reverse_Primer_Seq 421 397 703318O13_region_A3_193470_13_Forward_Primer_Seq 193444 193468 704318O13_region_A3_193470_13_Reverse_Primer_Seq 193589 193565 705318O13_region_A3_183305_14_Forward_Primer_Seq 183239 183263 706318O13_region_A3_183305_14_Reverse_Primer_Seq 183384 183360 707318O13_region_A3_55050_14_Forward_Primer_Seq 54998 55022 708318O13_region_A3_55050_14_Reverse_Primer_Seq 55144 55120 709318O13_region_A3_224693_21_Forward_Primer_Seq 224656 224682 710318O13_region_A3_224693_21_Reverse_Primer_Seq 224803 224779 711318O13_region_A3_207216_12_Forward_Primer_Seq 207152 207176 712318O13_region_A3_207216_12_Reverse_Primer_Seq 207299 207276 713318O13_region_A3_4654_22_Forward_Primer_Seq 4612 4636 714318O13_region_A3_4654_22_Reverse_Primer_Seq 4760 4736 715318O13_region_A3_408959_13_Forward_Primer_Seq 408918 408942 716318O13_region_A3_408959_13_Reverse_Primer_Seq 409066 409042 717318O13_region_A3_132288_22_Forward_Primer_Seq 132192 132216 718318O13_region_A3_132288_22_Reverse_Primer_Seq 132340 132316 719318O13_region_A3_292822_20_Forward_Primer_Seq 292747 292771 720318O13_region_A3_292822_20_Reverse_Primer_Seq 292895 292871 721318O13_region_A3_311076_12_Forward_Primer_Seq 311027 311051 722318O13_region_A3_311076_12_Reverse_Primer_Seq 311175 311152 723318O13_region_A3_509623_13_Forward_Primer_Seq 509584 509608 724318O13_region_A3_509623_13_Reverse_Primer_Seq 509732 509708 725318O13_region_A3_190404_14_Forward_Primer_Seq 190358 190382 726318O13_region_A3_190404_14_Reverse_Primer_Seq 190506 190482 727318O13_region_A3_164916_15_Forward_Primer_Seq 164808 164832 728318O13_region_A3_164916_15_Reverse_Primer_Seq 164957 164933 729318O13_region_A3_21028_13_Forward_Primer_Seq 21001 21026 730318O13_region_A3_21028_13_Reverse_Primer_Seq 21150 21126 731318O13_region_A3_208012_17_Forward_Primer_Seq 207955 207979 732318O13_region_A3_208012_17_Reverse_Primer_Seq 208104 208085 733318O13_region_A3_484089_14_Forward_Primer_Seq 484036 484060 734318O13_region_A3_484089_14_Reverse_Primer_Seq 484185 484161 735318O13_region_A3_332780_17_Forward_Primer_Seq 332723 332747 736318O13_region_A3_332780_17_Reverse_Primer_Seq 332872 332853 737318O13_region_A3_480137_37_Forward_Primer_Seq 480059 480084 738318O13_region_A3_480137_37_Reverse_Primer_Seq 480208 480182 739318O13_region_A3_441056_14_Forward_Primer_Seq 441011 441035 740318O13_region_A3_441056_14_Reverse_Primer_Seq 441161 441138 741318O13_region_A3_77486_11_Forward_Primer_Seq 77447 77471 742318O13_region_A3_77486_11_Reverse_Primer_Seq 77597 77573 743318O13_region_A3_272468_11_Forward_Primer_Seq 272423 272447 744318O13_region_A3_272468_11_Reverse_Primer_Seq 272573 272549 745318O13_region_A3_425319_17_Forward_Primer_Seq 425233 425257 746318O13_region_A3_425319_17_Reverse_Primer_Seq 425383 425359 747318O13_region_A3_413879_31_Forward_Primer_Seq 413835 413859 748318O13_region_A3_413879_31_Reverse_Primer_Seq 413985 413961 749318O13_region_A3_80477_64_Forward_Primer_Seq 80440 80464 750318O13_region_A3_80477_64_Reverse_Prime_Seq 80591 80567 751318O13_region_A3_277272_50_Forward_Primer_Seq 277213 277237 752318O13_region_A3_277272_50_Reverse_Primer_Seq 277364 277340 753318O13_region_A3_509642_13_Forward_Primer_Seq 509604 509628 754318O13_region_A3_509642_13_Reverse_Primer_Seq 509755 509731 755318O13_region_A3_321771_14_Forward_Primer_Seq 321663 321687 756318O13_region_A3_321771_14_Reverse_Primer_Seq 321815 321791 757318O13_region_A3_26788_12_Forward_Primer_Seq 26734 26758 758318O13_region_A3_26788_12_Reverse_Primer_Seq 26886 26862 759318O13_region_A3_262706_16_Forward_Primer_Seq 262649 262673 760318O13_region_A3_262706_16_Reverse_Primer_Seq 262802 262778 761318O13_region_A3_243928_16_Forward_Primer_Seq 243891 243915 762318O13_region_A3_243928_16_Reverse_Primer_Seq 244044 244020 763318O13_region_A3_23246_148_Forward_Primer_Seq 23215 23239 764318O13_region_A3_23246_148_Reverse_Primer_Seq 23368 23344 765318O13_region_A3_165406_12_Forward_Primer_Seq 165367 165391 766318O13_region_A3_165406_12_Reverse_Primer_Seq 165521 165497 767318O13_region_A3_486294_14_Forward_Primer_Seq 486208 486232 768318O13_region_A3_486294_14_Reverse_Primer_Seq 486362 486338 769318O13_region_A3_46754_12_Forward_Primer_Seq 46661 46685 770318O13_region_A3_46754_12_Reverse_Primer_Seq 46816 46792 771318O13_region_A3_381116_15_Forward_Primer_Seq 381080 381104 772318O13_region_A3_381116_15_Reverse_Primer_Seq 381235 381211 773318O13_region_A3_350369_11_Forward_Primer_Seq 350295 350319 774318O13_region_A3_350369_11_Reverse_Primer_Seq 350450 350426 775318O13_region_A3_138841_13_Forward_Primer_Seq 138795 138819 776318O13_region_A3_138841_13_Reverse_Primer_Seq 138950 138926 777318O13_region_A3_12158_142_Forward_Primer_Seq 12117 12141 778318O13_region_A3_12158_142_Reverse_Primer_Seq 12272 12248 779318O13_region_A3_315368_13_Forward_Primer_Seq 315310 315334 780318O13_region_A3_315368_13_Reverse_Primer_Seq 315465 315441 781318O13_region_A3_307549_13_Forward_Primer_Seq 307464 307488 782318O13_region_A3_307549_13_Reverse_Primer_Seq 307619 307595 783318O13_region_A3_159857_14_Forward_Primer_Seq 159772 159796 784318O13_region_A3_159857_14_Reverse_Primer_Seq 159928 159904 785318O13_region_A3_140551_15_Forward_Primer_Seq 140454 140478 786318O13_region_A3_140551_15_Reverse_Primer_Seq 140610 140586 787318O13_region_A3_279869_11_Forward_Primer_Seq 279797 279821 788318O13_region_A3_279869_11_Reverse_Primer_Seq 279953 279929 789318O13_region_A3_78292_35_Forward_Primer_Seq 78265 78291 790318O13_region_A3_78292_35_Reverse_Primer_Seq 78422 78397 791318O13_region_A3_185019_12_Forward_Primer_Seq 184953 184977 792318O13_region_A3_185019_12_Reverse_Primer_Seq 185111 185087 793318O13_region_A3_409164_13_Forward_Primer_Seq 409082 409106 794318O13_region_A3_409164_13_Reverse_Primer_Seq 409240 409219 795318O13_region_A3_75392_14_Forward_Primer_Seq 75287 75311 796318O13_region_A3_75392_14_Reverse_Primer_Seq 75445 75421 797318O13_region_A3_231320_12_Forward_Primer_Seq 231269 231293 798318O13_region_A3_231320_12_Reverse_Primer_Seq 231429 231405 799318O13_region_A3_381102_14_Forward_Primer_Seq 381041 381064 800318O13_region_A3_381102_14_Reverse_Primer_Seq 381201 381176 801318O13_region_A3_491826_15_Forward_Primer_Seq 491753 491777 802318O13_region_A3_491826_15_Reverse_Primer_Seq 491914 491891 803318O13_region_A3_56365_21_Forward_Primer_Seq 56336 56360 804318O13_region_A3_56365_21_Reverse_Primer_Seq 56497 56473 805318O13_region_A3_372628_15_Forward_Primer_Seq 372554 372578 806318O13_region_A3_372628_15_Reverse_Primer_Seq 372715 372691 807318O13_region_A3_217037_11_Forward_Primer_Seq 216919 216943 808318O13_region_A3_217037_11_Reverse_Primer_Seq 217081 217057 809318O13_region_A3_302609_11_Forward_Primer_Seq 302575 302599 810318O13_region_A3_302609_11_Reverse_Primer_Seq 302737 302713 811318O13_region_A3_341804_11_Forward_Primer_Seq 341686 341710 812318O13_region_A3_341804_11_Reverse_Primer_Seq 341848 341824 807318O13_region_A3_217037_11_Forward_Primer_Seq 216919 216943 808318O13_region_A3_217037_11_Reverse_Primer_Seq 217081 217057 813318O13_region_A3_264929_68_Forward_Primer_Seq 264862 264886 814318O13_region_A3_264929_68_Reverse_Primer_Seq 265024 265000 815318O13_region_A3_55499_12_Forward_Primer_Seq 55400 55424 816318O13_region_A3_55499_12_Reverse_Primer_Seq 55563 55539 817318O13_region_A3_295634_14_Forward_Primer_Seq 295538 295562 818318O13_region_A3_295634_14_Reverse_Primer_Seq 295702 295677 819318O13_region_A3_269358_15_Forward_Primer_Seq 269242 269266 820318O13_region_A3_269358_15_Reverse_Primer_Seq 269406 269382 821318O13_region_A3_457009_24_Forward_Primer_Seq 456924 456948 822318O13_region_A3_457009_24_Reverse_Primer_Seq 457088 457064 823318O13_region_A3_176598_14_Forward_Primer_Seq 176554 176578 824318O13_region_A3_176598_14_Reverse_Primer_Seq 176718 176694 825318O13_region_A3_278266_12_Forward_Primer_Seq 278210 278234 826318O13_region_A3_278266_12_Reverse_Primer_Seq 278376 278350 827318O13_region_A3_391810_12_Forward_Primer_Seq 391683 391707 828318O13_region_A3_391810_12_Reverse_Primer_Seq 391851 391826 829318O13_region_A3_269485_15_Forward_Primer_Seq 269383 269407 830318O13_region_A3_269485_15_Reverse_Primer_Seq 269551 269527 831318O13_region_A3_359247_17_Forward_Primer_Seq 359218 359243 832318O13_region_A3_359247_17_Reverse_Primer_Seq 359386 359362 833318O13_region_A3_315094_13_Forward_Primer_Seq 314976 315002 834318O13_region_A3_315094_13_Reverse_Primer_Seq 315145 315120 835318O13_region_A3_307823_13_Forward_Primer_Seq 307784 307809 836318O13_region_A3_307823_13_Reverse_Primer_Seq 307953 307927 837318O13_region_A3_248588_15_Forward_Primer_Seq 248540 248564 838318O13_region_A3_248588_15_Reverse_Primer_Seq 248709 248684 839318O13_region_A3_252426_85_Forward_Primer_Seq 252398 252423 840318O13_region_A3_252426_85_Reverse_Primer_Seq 252568 252543 841318O13_region_A3_513314_16_Forward_Primer_Seq 513209 513233 842318O13_region_A3_513314_16_Reverse_Primer_Seq 513379 513355 843318O13_region_A3_68183_14_Forward_Primer_Seq 68108 68132 844318O13_region_A3_68183_14_Reverse_Primer_Seq 68279 68255 845318O13_region_A3_471191_13_Forward_Primer_Seq 471059 471083 846318O13_region_A3_471191_13_Reverse_Primer_Seq 471231 471206 847318O13_region_A3_163547_18_Forward_Primer_Seq 163459 163483 848318O13_region_A3_163547_18_Reverse_Primer_Seq 163632 163608 849318O13_region_A3_417867_15_Forward_Primer_Seq 417839 417863 850318O13_region_A3_417867_15_Reverse_Primer_Seq 418014 417990 851318O13_region_A3_332465_14_Forward_Primer_Seq 332346 332370 852318O13_region_A3_332465_14_Reverse_Primer_Seq 332523 332499 853318O13_region_A3_207697_14_Forward_Primer_Seq 207578 207602 854318O13_region_A3_207697_14_Reverse_Primer_Seq 207755 207731 855318O13_region_A3_277229_43_Forward_Primer_Seq 277186 277210 856318O13_region_A3_277229_43_Reverse_Primer_Seq 277364 277340 857318O13_region_A3_36366_11_Forward_Primer_Seq 36323 36345 858318O13_region_A3_36366_11_Reverse_Primer_Seq 36501 36477 859318O13_region_A3_91970_12_Forward_Primer_Seq 91938 91962 860318O13_region_A3_91970_12_Reverse_Primer_Seq 92116 92091 861318O13_region_A3_211533_11_Forward_Primer_Seq 211406 211430 862318O13_region_A3_211533_11_Reverse_Primer_Seq 211585 211561 863318O13_region_A3_336301_11_Forward_Primer_Seq 336174 336198 864318O13_region_A3_336301_11_Reverse_Primer_Seq 336353 336329 865318O13_region_A3_441603_14_Forward_Primer_Seq 441539 441563 866318O13_region_A3_441603_14_Reverse_Primer_Seq 441718 441694 867318O13_region_A3_468354_15_Forward_Primer_Seq 468263 468287 868318O13_region_A3_468354_15_Reverse_Primer_Seq 468442 468418 869318O13_region_A3_188983_18_Forward_Primer_Seq 188855 188879 870318O13_region_A3_188983_18_Reverse_Primer_Seq 189035 189009 871318O13_region_A3_115502_17_Forward_Primer_Seq 115469 115493 872318O13_region_A3_115502_17_Reverse_Primer_Seq 115649 115625 873318O13_region_A3_163006_13_Forward_Primer_Seq 162972 162996 874318O13_region_A3_163006_13_Reverse_Primer_Seq 163153 163129 875318O13_region_A3_119283_14_Forward_Primer_Seq 119199 119224 876318O13_region_A3_119283_14_Reverse_Primer_Seq 119381 119357 877318O13_region_A3_491126_11_Forward_Primer_Seq 491062 491086 878318O13_region_A3_491126_11_Reverse_Primer_Seq 491244 491220 879318O13_region_A3_99512_21_Forward_Primer_Seq 99398 99422 880318O13_region_A3_99512_21_Reverse_Primer_Seq 99581 99557 881318O13_region_A3_280291_17_Forward_Primer_Seq 280201 280226 882318O13_region_A3_280291_17_Reverse_Primer_Seq 280385 280361 883318O13_region_A3_138443_19_Forward_Primer_Seq 138304 138329 884318O13_region_A3_138443_19_Reverse_Primer_Seq 138488 138465 885318O13_region_A3_115973_14_Forward_Primer_Seq 115832 115856 886318O13_region_A3_115973_14_Reverse_Primer_Seq 116016 115992 887318O13_region_A3_329977_14_Forward_Primer_Seq 329864 329889 888318O13_region_A3_329977_14_Reverse_Primer_Seq 330050 330026 889318O13_region_A3_205203_14_Forward_Primer_Seq 205090 205115 890318O13_region_A3_205203_14_Reverse_Primer_Seq 205276 205252 891318O13_region_A3_153114_12_Forward_Primer_Seq 152969 152993 892318O13_region_A3_153114_12_Reverse_Primer_Seq 153156 153132 893318O13_region_A3_34581_13_Forward_Primer_Seq 34523 34547 894318O13_region_A3_34581_13_Reverse_Primer_Seq 34712 34688 895318O13_region_A3_292577_19_Forward_Primer_Seq 292549 292573 896318O13_region_A3_292577_19_Reverse_Primer_Seq 292739 292715 897318O13_region_A3_445391_20_Forward_Primer_Seq 445356 445382 898318O13_region_A3_445391_20_Reverse_Primer_Seq 445547 445523 899318O13_region_A3_350540_17_Forward_Primer_Seq 350421 350445 900318O13_region_A3_350540_17_Reverse_Primer_Seq 350612 350588 901318O13_region_A3_453879_15_Forward_Primer_Seq 453725 453750 902318O13_region_A3_453879_15_Reverse_Primer_Seq 453918 453894 903318O13_region_A3_201246_13_Forward_Primer_Seq 201128 201153 904318O13_region_A3_201246_13_Reverse_Primer_Seq 201321 201297 905318O13_region_A3_326020_13_Forward_Primer_Seq 325902 325927 906318O13_region_A3_326020_13_Reverse_Primer_Seq 326095 326071 907318O13_region_A3_503801_14_Forward_Primer_Seq 503656 503680 908318O13_region_A3_503801_14_Reverse_Primer_Seq 503849 503823 909318O13_region_A3_302400_52_Forward_Primer_Seq 302283 302307 910318O13_region_A3_302400_52_Reverse_Primer_Seq 302481 302456 911318O13_region_A3_448857_15_Forward_Primer_Seq 448748 448772 912318O13_region_A3_448857_15_Reverse_Primer_Seq 448947 448924 913318O13_region_A3_48364_14_Forward_Primer_Seq 48232 48256 914318O13_region_A3_48364_14_Reverse_Primer_Seq 48435 48412 915318O13_region_A3_251804_48_Forward_Primer_Seq 251738 251762 916318O13_region_A3_251804_48_Reverse_Primer_Seq 251942 251918 917318O13_region_A3_382583_13_Forward_Primer_Seq 382549 382574 918318O13_region_A3_382583_13_Reverse_Primer_Seq 382753 382728 919318O13_region_A3_124737_14_Forward_Primer_Seq 124641 124665 920318O13_region_A3_124737_14_Reverse_Primer_Seq 124846 124822 921318O13_region_A3_124766_13_Forward_Primer_Seq 124641 124665 922318O13_region_A3_124766_13_Reverse_Primer_Seq 124846 124822 923318O13_region_A3_461351_16_Forward_Primer_Seq 461218 461242 924318O13_region_A3_461351_16_Reverse_Primer_Seq 461426 461402 925318O13_region_A3_64953_19_Forward_Primer_Seq 64798 64823 926318O13_region_A3_64953_19_Reverse_Primer_Seq 65011 64987 927318O13_region_A3_366586_13_Forward_Primer_Seq 366508 366532 928318O13_region_A3_366586_13_Reverse_Primer_Seq 366722 366698 929318O13_region_A3_46190_15_Forward_Primer_Seq 46012 46037 930318O13_region_A3_46190_15_Reverse_Primer_Seq 46228 46205 931318O13_region_A3_81016_11_Forward_Primer_Seq 80927 80952 932318O13_region_A3_81016_11_Reverse_Primer_Seq 81146 81122 933318O13_region_A3_134426_14_Forward_Primer_Seq 134253 134277 934318O13_region_A3_134426_14_Reverse_Primer_Seq 134474 134449 935318O13_region_A3_292724_14_Forward_Primer_Seq 292549 292573 936318O13_region_A3_292724_14_Reverse_Primer_Seq 292771 292747 937318O13_region_A3_187096_17_Forward_Primer_Seq 187058 187082 938318O13_region_A3_187096_17_Reverse_Primer_Seq 187282 187257 939318O13_region_A3_381693_13_Forward_Primer_Seq 381658 381683 940318O13_region_A3_381693_13_Reverse_Primer_Seq 381885 381863 941318O13_region_A3_361286_33_Forward_Primer_Seq 361173 361197 942318O13_region_A3_361286_33_Reverse_Primer_Seq 361401 361376 943318O13_region_A3_482668_14_Forward_Primer_Seq 482592 482616 944318O13_region_A3_482668_14_Reverse_Primer_Seq 482821 482796 945318O13_region_A3_128002_12_Forward_Primer_Seq 127882 127906 946318O13_region_A3_128002_12_Reverse_Primer_Seq 128112 128087 947318O13_region_A3_499270_14_Forward_Primer_Seq 499184 499208 948318O13_region_A3_499270_14_Reverse_Primer_Seq 499422 499398 949318O13_region_A3_231650_12_Forward_Primer_Seq 231568 231592 950318O13_region_A3_231650_12_Reverse_Primer_Seq 231809 231788 951318O13_region_A3_199851_13_Forward_Primer_Seq 199762 199786 952318O13_region_A3_199851_13_Reverse_Primer_Seq 200012 199988 953318O13_region_A3_324629_13_Forward_Primer_Seq 324540 324564 954318O13_region_A3_324629_13_Reverse_Primer_Seq 324790 324766 955318O13_region_A3_374190_19_Forward_Primer_Seq 374129 374152 956318O13_region_A3_374190_19_Reverse_Primer_Seq 374394 374370 957318O13_region_A3_460603_13_Forward_Primer_Seq 460450 460474 958318O13_region_A3_460603_13_Reverse_Primer_Seq 460715 460691 959318O13_region_A3_108681_14_Forward_Primer_Seq 108524 108548 960318O13_region_A3_108681_14_Reverse_Primer_Seq 108791 108768 961318O13_region_A3_459791_47_Forward_Primer_Seq 459639 459663 962318O13_region_A3_459791_47_Reverse_Primer_Seq 459907 459883 963318O13_region_A3_4257_20_Forward_Primer_Seq 4172 4196 964318O13_region_A3_4257_20_Reverse_Primer_Seq 4450 4425 965318O13_region_A3_238810_14_Forward_Primer_Seq 238563 238589 966318O13_region_A3_238810_14_Reverse_Primer_Seq 238850 238826 967318O13_region_A3_245817_14_Forward_Primer_Seq 245713 245738 968318O13_region_A3_245817_14_Reverse_Primer_Seq 246001 245977 969318O13_region_A3_245956_14_Forward_Primer_Seq 245713 245738 970318O13_region_A3_245956_14_Reverse_Primer_Seq 246001 245977 971318O13_region_A3_74148_14_Forward_Primer_Seq 74050 74075 972318O13_region_A3_74148_14_Reverse_Primer_Seq 74338 74314 973318O13_region_A3_74089_15_Forward_Primer_Seq 74050 74075 974318O13_region_A3_74089_15_Reverse_Primer_Seq 74338 74314 975318O13_region_A3_241686_12_Forward_Primer_Seq 241470 241494 976318O13_region_A3_241686_12_Reverse_Primer_Seq 241765 241741 977318O13_region_A3_47476_12_Forward_Primer_Seq 47280 47304 978318O13_region_A3_47476_127_Reverse_Primer_Seq 47577 47554 979318O13_region_A3_164550_12_Forward_Primer_Seq 164323 164347 980318O13_region_A3_164550_12_Reverse_Primer_Seq 164621 164598 981318O13_region_A3_101255_15_Forward_Primer_Seq 101119 101144 982318O13_region_A3_101255_15_Reverse_Primer_Seq 101418 101392 983515O02_region_G2_16189_11_Forward_Primer 16144 16168 984515O02_region_G2_16189_11_Reverse_Primer 16244 16220 985515O02_region_G2_71925_13_Forward_Primer 71880 71905 986515O02_region_G2_71925_13_Reverse_Primer 71987 71963 987515O02_region_G2_4707_12_Forward_Primer 4660 4684 988515O02_region_G2_4707_12_Reverse_Primer 4769 4743 989515O02_region_G2_118904_18_Forward_Primer 118847 118871 990515O02_region_G2_118904_18_Reverse_Primer 118957 118932 991515O02_region_G2_13655_17_Forward_Primer 13567 13592 992515O02_region_G2_13655_17_Reverse_Primer 13698 13673 993515O02_region_G2_53900_13_Forward_Primer 53843 53867 994515O02_region_G2_53900_13_Reverse_Primer 53985 53961 995515O02_region_G2_8079_14_Forward_Primer 8023 8047 996515O02_region_G2_8079_14_Reverse_Primer 8167 8143 997515O02_region_G2_9969_28_Forward_Primer 9917 9941 998515O02_region_G2_9969_28_Reverse_Primer 10062 10038 999515O02_region_G2_72308_77_Forward_Primer 72272 72298 1000515O02_region_G2_72308_77_Reverse_Primer 10062 10038 1001515O02_region_G2_99475_19_Forward_Primer 99408 99433 1002515O02_region_G2_99475_19_Reverse_Primer 99554 99530 1003515O02_region_G2_118615_18_Forward_Primer 118512 118535 1004515O02_region_G2_118615_18_Reverse_Primer 118658 118634 1005515O02_region_G2_119001_46_Forward_Primer 118931 118956 1006515O02_region_G2_119001_46_Reverse_Primer 119079 119055 1007515O02_region_G2_118958_43_Forward_Primer 118931 118956 1008515O02_region_G2_118958_43_Reverse_Primer 119079 119055 1009515O02_region_G2_17197_13_Forward_Primer 17128 17152 1010515O02_region_G2_17197_13_Reverse_Primer 17276 17252 1011515O02_region_G2_105163_29_Forward_Primer 105068 105092 1012515O02_region_G2_105163_29_Reverse_Primer 105217 105192 1013515O02_region_G2_111335_13_Forward_Primer 111308 111332 1014515O02_region_G2_111335_13_Reverse_Primer 111458 111434 1015515O02_region_G2_106396_13_Forward_Primer 106318 106342 1016515O02_region_G2_106396_13_Reverse_Primer 106469 106445 1017515O02_region_G2_59229_17_Forward_Primer 59203 59227 1018515O02_region_G2_59229_17_Reverse_Primer 59354 59330 1019515O02_region_G2_73795_20_Forward_Primer 73769 73793 1020515O02_region_G2_73795_20_Reverse_Primer 73921 73896 1021515O02_region_G2_85664_20_Forward_Primer 85586 85611 1022515O02_region_G2_85664_20_Reverse_Primer 85738 85714 1023515O02_region_G2_36921_17_Forward_Primer 36830 36854 1024515O02_region_G2_36921_17_Reverse_Primer 36983 36959 1025515O02_region_G2_124150_19_Forward_Primer 124073 124096 1026515O02_region_G2_124150_19_Reverse_Primer 124227 124203 1027515O02_region_G2_5089_14_Forward_Primer 4999 5024 1028515O02_region_G2_5089_14_Reverse_Primer 5156 5132 1029515O02_region_G2_58221_15_Forward_Primer 58197 58220 1030515O02_region_G2_58221_15_Reverse_Primer 58354 58330 1031515O02_region_G2_96139_14_Forward_Primer 96022 96046 1032515O02_region_G2_96139_14_Reverse_Primer 96182 96158 1033515O02_region_G2_70595_13_Forward_Primer 70472 70496 1034515O02_region_G2_70595_13_Reverse_Primer 70634 70608 1035515O02_region_G2_4340_15_Forward_Primer 4312 4337 1036515O02_region_G2_4340_15_Reverse_Primer 4477 4454 1037515O02_region_G2_90417_11_Forward_Primer 90335 90359 1038515O02_region_G2_90417_11_Reverse_Primer 90503 90479 1039515O02_region_G2_49711_17_Forward_Primer 49652 49676 1040515O02_region_G2_49711_17_Reverse_Primer 49820 49796 1041515O02_region_G2_63053_13_Forward_Primer 63005 63029 1042515O02_region_G2_63053_13_Reverse_Primer 63173 63148 1043515O02_region_G2_63076_14_Forward_Primer 63005 63029 1044515O02_region_G2_63076_14_Reverse_Primer 63173 63148 1045515O02_region_G2_44442_12_Forward_Primer 44335 44359 1046515O02_region_G2_44442_12_Reverse_Primer 44505 44481 1047515O02_region_G2_44422_19_Forward_Primer 44335 44359 1048515O02_region_G2_44422_19_Reverse_Primer 44505 44481 1049515O02_region_G2_44158_19_Forward_Primer 44075 44100 1050515O02_region_G2_44158_19_Reverse_Primer 44252 44227 1051515O02_region_G2_44141_17_Forward_Primer 44075 44100 1052515O02_region_G2_44141_17_Reverse_Primer 44252 44227 1053515O02_region_G2_90762_17_Forward_Primer 90637 90663 1054515O02_region_G2_90762_17_Reverse_Primer 90814 90790 1055515O02_region_G2_106241_14_Forward_Primer 106160 106184 1056515O02_region_G2_106241_14_Reverse_Primer 106341 106317 1057515O02_region_G2_109676_12_Forward_Primer 109609 109634 1058515O02_region_G3_109676_12_Reverse_Primer 109793 109768 1059515O02_region_G2_86242_14_Forward_Primer 86134 86158 1060515O02_region_G2_86242_14_Reverse_Primer 86318 86293 1061515O02_region_G2_83109_12_Forward_Primer 83017 83041 1062515O02_region_G2_83109_12_Reverse_Primer 83202 83178 1063515O02_region_G2_10461_15_Forward_Primer 10418 10442 1064515O02_region_G2_10461_15_Reverse_Primer 10609 10585 1065515O02_region_G2_67608_15_Forward_Primer 67552 67577 1066515O02_region_G2_67608_15_Reverse_Primer 67745 67721 1067515O02_region_G2_63275_46_Forward_Primer 63148 63173 1068515O02_region_G2_63275_46_Reverse_Primer 63347 63323 1069515O02_region_G2_62405_14_Forward_Primer 62374 62399 1070515O02_region_G2_62405_14_Reverse_Primer 62576 62552 1071515O02_region_G2_33563_12_Forward_Primer 33460 33484 1072515O02_region_G2_33563_12_Reverse_Primer 33670 33646 1073515O02_region_G2_33146_14_Forward_Primer 32949 32973 1074515O02_region_G2_33146_14_Reverse_Primer 33191 33167 1075515O02_region_G2_102179_29_Forward_Primer 102102 102126 1076515O02_region_G2_102179_29_Reverse_Primer 102352 102327 1077515O02_region_G2_2646_15_Forward_Primer 2553 2577 1078515O02_region_G2_2646_15_Reverse_Primer 2809 2784 1079515O02_region_G2_76652_24_Forward_Primer 76567 76591 1080515O02_region_G2_76652_24_Reverse_Primer 76835 76812 1081515O02_region_G2_66280_14_Forward_Primer 66052 66077 1082515O02_region_G2_66280_14_Reverse_Primer 66334 66309 1083515O02_region_G2_54768_13_Forward_Primer 54640 54666 1084515O02_region_G2_54768_13_Reverse_Primer 54923 54899 1085515O02_region_G2_62580_14_Forward_Primer 62552 62576 1086515O02_region_G2_62580_14_Reverse_Primer 62840 62816 1087515O02_region_G2_34598_55_Forward_Primer 34473 34497 1088515O02_region_G2_34598_55_Reverse_Primer 34765 34739 1089515O02_region_G2_77680_13_Forward_Primer 77444 77470 1090515O02_region_G2_77680_13_Reverse_Primer 77741 77716 1091515O02_region_G2_77693_12_Forward_Primer 77444 77470 1092515O02_region_G2_77693_12_Reverse_Primer 77741 77716 1093515O02_region_G2_97392_14_Forward_Primer 97255 97280 1094515O02_region_G2_97392_14_Reverse_Primer 97554 97530 1095515O02_region_G2_97359_15_Forward_Primer 97255 97280 1096515O02_region_G2_97359_15_Reverse_Primer 97554 97530 1120 consensusLRR1121 rhg1LRR 1122 Rhg4LRR Primer location on 240O17_region_G3 1123240O17_region_G3_forward_1_b 45046-45072

DETAILED DESCRIPTION OF THE INVENTION

A) rhg1

The present invention provides a method for the production of a soybeanplant having an rhg1 SCN resistant allele comprising: (A) crossing afirst soybean plant having an rhg1 SCN rersistant allele with a secondsoybean plant having an rhg1 SCN sensitive allele to produce asegregating population; (B) screening the segregating population for amember having an rhg1 SCN resistant allele with a first nucleic acidmolecule capable of specifically hybridizing to linkage group G, whereinthe first nucleic acid molecule specifically hybridizes to a secondnucleic acid molecule that is linked to the rhg1 SCN resistant allele;and, (C) selecting the member for further crossing and selection.

rhg1 is located on linkage group G (Concibido et al., Crop Sci.36:1643-1650 (1996)). SCN resistant alleles of rhg1 provide partialresistance to SCN races 1, 2, 3, 5, 6, and 14 (Concibido et al. (CropSci. 37:258-264 (1997)). Also, Webb (U.S. Pat. No. 5,491,081) reportsthat a QTL on linkage group G (rhg1) provides partial resistance to SCNraces 1, 2, 3, 5, and 14. rhg1 and Rhg4 provide complete or nearlycomplete resistance to SCN race 3 (U.S. Pat. No. 5,491,081). Whileinitially thought to be a recessive gene, rhg1 classification as arecessive gene has been questioned.

Using bioinformatic approaches, the rhg1 coding region is predicted tocontain either four exons (rhg1, v.1)(coding coordinates 45163-45314,45450-45509, 46941-48763, and 48975-49573 of SEQ ID NO: 2) or two exons(rhg1, v.2) (coding coordinates 46798-48763 and 48975-49573 of SEQ IDNO: 3). rhg1, v.1 encodes an 877 amino acid polypeptide. rhg1, v.2encodes an 854 amino acid length polypeptide. rhg1 codes for a Xa21-likereceptor kinase (SEQ ID NOs: 1097, 1098, and 1100-1115) (Song, et al.,Science 270, 1804-1806 (1995)). rhg1 has an extracellular leucine richrepeat (LRR) domain (rhg1, v.1, SEQ ID NO: 1097, residues 164-457; rhg1,v.2, SEQ ID NO: 1098, residues 141-434), a transmembrane domain (rhg1,v.1, SEQ ID NO: 1097, residues 508-530; rhg1, v.2, SEQ ID NO: 1098,residues 33-51, and 485-507) and serine/threonine protein kinase (STK)domain (rhg1, v.1, SEQ ID NO: 1097, residues 578-869; rhg1, v.2, SEQ IDNO: 1098, residues 555-846). In a preferred embodiment, the LRR domainhas multiple LRR repeats. In a more preferred embodiment, the LRR domainhas 12 LRR H repeats.

To identify proteins similar to the proteins encoded by rhg1 candidates,database searches are performed using the predicted peptide sequences.The rhg1 candidate shows similarity to CAA18124, which is theArabidopsis putative receptor kinase (58.4% similarity and 35.8%identity, (CLUSTALW (default parameters), Thompson et al., Nucleic AcidsRes. 22:4673-4680 (1994)), GCG package, Genetics Computer Group,Madison, Wis.), and the apple leucine-rich receptor-like protein kinase(g3641252) (53.2% similarity and 31.5% identity, (CLUSTALW (defaultparameters))), which has both LLR and STK domains, showing conservationin both the LLR and STK domains. The predicted LRR extracellular domainshows similarity to the tomato resistance genes Cf-2.1 (Lycopersiconpimpinellifolium) (66.9% similarity and 45.4% identity (CLUSTALW(default parameters))) and Cf-2.2 (Lycopersicon pimpinellifolium) (66.9%similarity and 45.4% identity (CLUSTALW (default parameters))).

FIG. 1 is an alignment of the LRR domain of the rhg1 gene. A consensussequence for the LRR is shown as the top row of the alignment. Each rowof amino acids represents an LRR domain. The boxed region indicates theputative β-turn/β-sheet structural motif postulated to be involved inligand binding (Jones and Jones, Adv. Bot. Res. Incorp. Adv. Plant Path.24; 89-167 (1997)). The hydrophobic leucine residues are thought toproject into the core of the protein while the flanking amino acids arethought to be solvent exposed where they may interact with the ligand(Kobe and Deisenhofer, Nature 374; 183-186 (1995)). Non-conservativechanges in this region are thought to affect folding. An “x” representsan arbitrary amino acid while an “a” represents a hydrophobic residue(leucine, isoleucine, methionine, valine, or phenylalanine). Amino acidsubstitutions between resistant and sensitive phenotypes are bordered bya double line. The amino acid substitution within the 302-325 region isa histidine/asparagine substitution, and the amino acid substitutionwithin the 422-445 region is a phenylalanine/serine substitution.

As used herein, a naturally occurring rhg1 allele is any allele thatencodes for a protein having an extracellular LRR, a transmembranedomain, and STK domain where the naturally occurring allele is presenton linkage group G and where certain rhg1 alleles, but not all rhg1alleles, are capable of providing or contributing to resistance orpartial resistance to a race of SCN. It is understood that such anallele can, using for example methods disclosed herein, be manipulatedso that the nucleic acid molecule encoding the protein is no longerpresent on linkage group G. It is also understood that such an allelecan, using for example methods disclosed herein, be manipulated so thatthe nucleic acid molecule sequence is altered.

As used herein, an rhg1 SCN resistant allele is any rhg1 allele wherethat allele alone or in combination with other SCN resistant allelespresent in the plant, such as an Rhg4 SCN resistant allele, providesresistance to a race of SCN, and that resistance is due, at least inpart, to the genetic contribution of the rhg1 allele.

SCN resistance or partial resistance is determined by a comparison ofthe plant in question with a known SCN sensitive host, Lee 74, accordingto the method set forth in Schmitt, J. Nematol. 20:392-395 (1988). Asused herein, resistance to a particular race of SCN is defined as havingless than 10% of cyst development relative to the SCN sensitive host Lee74. Moreover, as used herein, partial resistance to a particular race ofSCN is defined as having more than 10% but less than 75% of cystdevelopment relative to the SCN sensitive host Lee 74.

Any soybean plant having an rhg1 SCN resistant allele can be used inconjunction with the present invention. Soybeans with known rhg1 SCNresistant alleles can be used. Such soybeans include but are not limitedto PI548402 (Peking), PI200499, A2869, Jack, A2069, PI209332 (No:4),PI404166 (Krasnoaarmejkaja), PI404198 (Sun huan do), PI437654(Er-hej-jan), PI438489 (Chiquita), PI507354 (Tokei 421), PI548655(Forrest), PI548988 (Pickett), PI84751, PI437654, PI40792, Pyramid,Nathan, AG2201, A3469, AG3901, A3904, AG4301, AG4401, AG4501, AG4601,PION9492, PI88788, Dyer, Custer, Manokin, and Doles. In a preferredaspect, the soybean plant having an rhg1 SCN resistant allele is an rhg1haplotype 2 allele. Examples of soybeans with an rhg1 haplotype 2 alleleare PI548402 (Peking), PI404166 (Krasnoaarmejkaja), PI404198 (Sun huando), PI437654 (Er-hejjan), PI438489 (Chiquita), PI507354 (Tokei 421),PI548655 (Forrest), PI548988 (Pickett), PI84751, PI437654, and PI40792.In addition, using the methods or agents of the present invention,soybeans and wild relative of soybean such as Glycine soja can bescreened for the presence of rhg1 SCN resistant alleles.

Any soybean plant having an rhg1 SCN sensitive allele can be used inconjunction with the present invention. Such soybeans include A3244,A2833, AG3001, Williams, Will, A2704, Noir, DK23-51, Lee 74, Essex,Minsoy, A1923, and Hutcheson. In a preferred aspect, the soybean planthaving an rhg1 SCN sensitive allele is an rhg1 A3244 allele. Inaddition, using the methods or agents of the present invention, soybeansand wild relatives of soybean such as Glycine soja can be screened forthe presence of rhg1 SCN sensitive alleles.

Table 2, below, is a table showing single nucleotide polymorphisms(SNPs) and insertions/deletions (INDEL) sites for eight haplotypesequences of rhg1. TABLE 2 Identification Base number of contig240O17_region_G3 of reference line A3244 Hap PI# Line Ph 45173 4530945400 45416 45439 45611 45916 45958 46049 46113 1 — A3244 S G G A T A AA A C A 2 PI548402 Peking R G A C C T A G A T G 3 PI423871 Toyosuzu — GA A T A A G A T G 4 PI518672 Will S G G A T A A A A C A 5 — A2704 S G GA T A A A A C A 6 PI290136 Noir S A A A C T G A T T A 7 PI548658 Lee 74S A A A C T G A T T A 8 PI200499 — R G A A C A A A A T A N/A PI548667Essex S A A A C T G A T T A N/A PI548389 Minsoy S G G A T A A A A C AN/A PI360843 Oshima. — — — — — — — — — — — N/A — A2869 R — — — — — — — —— — N/A PI540556 Jack R — — — — — — — — — — N/A — A2069 R — — — — — — —— — — N/A PI209332 No. 4 R — — — — — — — — — — Identification Basenumber of contig 240O17_region_G3 of reference line A3244 Hap PI# LinePh 46227 46703 47057 47140 47208 47571 47617 47796 47856 47937 1 — A3244S d1 0 T C C G C A T T 2 PI548402 Peking R 0 d2 C C C G C C C C 3PI423871 Toyosuzu — 0 0 T C C G C C C C 4 PI518672 Will S d1 0 T A T G CA T T 5 — A2704 S d1 0 T A T G C A T T 6 PI290136 Noir S 0 d14 T C C A AC C C 7 PI548658 Lee 74 S 0 d14 T C C G A C C C 8 PI200499 — R 0 d14 T CC G A C C C N/A PI548667 Essex S 0 d14 T C C G A C C C N/A PI548389Minsoy S d1 0 T A T G C A T T N/A PI360843 Oshima. — — 0 T A T G C A T/CT/C N/A — A2869 R 0 d14 T C C G A C C C N/A PI540556 Jack R — — — — — —— C C C N/A — A2069 R — — — — — — — C T/C T/C N/A PI209332 No. 4 R — — —— — — — C C C Identification Base number of contig 240O17_region_G3 ofreference line A3244 Hap PI# Line Ph 48012 48060 48073 48135 48279 4841348681 48881 49012 49316 1 — A3244 S T C C A C G A 0 A T 2 PI548402Peking R T C C G C G G d19 G T 3 PI423871 Toyosuzu — T C C G C G A 0 A T4 PI518672 Will S T C C A C G A 0 A T 5 — A2704 S C T T G T C — 0 G C 6PI290136 Noir S C T T G T C G 0 G C 7 PI548658 Lee 74 S C T T G T C G 0G C 8 PI200499 — R C T T G T C G 0 G C N/A PI548667 Essex S C T T G T CA/G 0 G C N/A PI548389 Minsoy S C/T C/T C/T A C G A 0 A T N/A PI360843Oshima. — T C C A/G C G A 0 A T N/A — A2869 R C T T G T C G 0 G C N/API540556 Jack R C T T G T C G 0 G C N/A — A2069 R C T T A T C G 0 G CN/A PI209332 No. 4 R C T T A/G T C G 0 G C

In Table 2, discrete haplotypes are designated 1 through 8. N/A refersto a haplotype that is not characterized. The Plant Introductionclassification number is indicated in the “PI#” column. A dash indicatesthat no PI number is known or assigned for the line under investigation.The line from which the sequences are derived is indicated in the “line”column, with a dash indicating an unknown or unnamed line. The “Ph.”(phenotype) column of table 2 indicates whether a given line has beenreported as resistant (R) to at least one race of SCN or sensitive (S).

The nucleotide bases located at each of 30 positions in each of thehaplotype sequences is shown in the columns labeled “Base number ofcontig 240O017-region-G3 of reference line A3244.” The base number atthe top of each column corresponds to the base number in contig240O17_region_G3 of reference line A3224 (SEQ ID NOs: 2 and 3). Theletters 0, A, C, and T correspond to the bases guanine, adenine,cytosine, and thymine. Two bases separated by a slash (A/G, C/T, or TIC)indicate uncertainty at the specified position of the haplotypesequence. A “d” followed by a number indicates a deletion of a thelength specified. That is, d1 is a one base deletion, d2 is a two basedeletion, d14 is a fourteen base deletion, and d19 is a nineteen basedeletion. A zero (0) indicates no deletion. A dash indicates that theidentity of the base is undetermined.

Examination of table 2 reveals that the amino acid substitutions in therhg1 coding region are common to the resistant lines PI467312(Cha-mo-shi-dou), PI88788 and the southern susceptible lines Essex,Hutchenson, Noir and A1923. As used herein, a “southern” cultivar is anycultivar from maturity groups VI, VII, VIII, IX, or X, and a “northern”cultivar is any cultivar from maturity groups 000, 00, 0, I, II, III,IV, or V. This data is consistent with the mapping experiments of Qui etal. (Theor Appl Genet 98:356-364 (1999)). Based on analysis of 200F_(2:3) families derived from a cross between Peking and Essex, theauthors failed to detect any significant association with SCN resistanceto races 1, 2, and 3, and the rhg1 locus on linkage group G. The authorspoint out that one of the markers, Bng122, which has been shown to havesignificant linkage to rhg1 (Concibido et al., Crop Sci. 36:1643-1650(1996)), is not polymorphic in the population employed. It is alsopossible that the susceptible southern lines contain rhg1 and thesusceptible phenotype reflects the polygenic nature of SCN resistance.In a study to uncover QTLs for sudden death syndrome (SDS) in soybean,two SCN resistant alleles originating from the susceptible parent Essexhave been described (Hnetkovsky et al., Crop Sci. 36:393-400).

Tables 3a, 3b, and 3c, below, show lines that share an rhg1 haplotype.TABLE 3a Haplotype 2 Lines PI# Line Ph. PI548402 Peking R PI404166Krasnoaarmejkaja R PI404198 (Sun huan do) R PI437654 Er-hej-jan RPI438489 (Chiquita) R PI507354 Tokei 421 R PI548655 Forrest R PI548988Pickett R PI84751 — R PI437654 — R PI40792 — —

TABLE 3b Haplotype 4 Lines PI# Line Ph. — Will S PI467312 Cha-mo-shi-douR PI88788 — R

TABLE 3c Haplotype 6 Lines PI# Line Ph. — Noir S — A1923 S — Hutcheson S

In Tables 3a, 3b, and 3c, Plant Introduction classification number isindicated in the “PI#” column. A dash indicates that no PI number isknown or assigned for the line in question. The line from which thesequences are derived is indicated in the “line” column, with a dashindicating an unknown or unnamed line. The “Ph.” column indicateswhether a given line has been reported as resistant (R) to at least onerace of SCN or sensitive (S), with a dash indicating that the phenotypeis unknown.

In a preferred aspect, the source of either an rhg1 SCN sensitive alleleor an rhg1 SCN resistant allele, or more preferably both, is an eliteplant. An “elite line” is any line that has resulted from breeding andselection for superior agronomic performance. Examples of elite linesare lines that are commercially available to farmers or soybean breederssuch as HARTZ™ variety H4994, HARTZ™ variety H5218, HARTZ™ varietyH5350, HARTZ™ variety H5545, HARTZ™ variety H5050, HARTZ™ variety H5454,HARTZ™ variety H5233, HARTZ™ variety H5488, HARTZ™ variety HLA572,HARTZ™ variety H6200, HARTZ™ variety H6104, HARTZ™ variety H6255, HARTZ™variety H6586, HARTZ™ variety H6191, HARTZ™ variety H7440, HARTZ™variety H4452 Roundup Ready™, HARTZ™ variety H4994 Roundup Ready™,HARTZ™ variety H4988 Roundup Ready™, HARTZ™ variety H5000 RoundupReady™, HARTZ™ variety H5147 Roundup Ready™, HARTZ™ variety H5247Roundup Ready™, HARTZ™ variety H5350 Roundup Ready™, HARTZ™ varietyH5545 Roundup Ready, HARTZ™ variety H5855 Roundup Ready™, HARTZ™ varietyH5088 Roundup Ready™, HARTZ™ variety H5164 Roundup Ready™, HARTZ™variety H5361 Roundup Ready™, HARTZ™ variety H5566 Roundup Ready™,HARTZ™ variety H5181 Roundup Ready™, HARTZ™ variety H5889 Roundup Ready,HARTZ™ variety H5999 Roundup Ready™, HARTZ™ variety H6013 RoundupReady™, HARTZ™ variety H6255 Roundup Ready™, HARTZ™ variety H6454Roundup Ready™, HARTZ™ variety H6686 Roundup Ready™, HARTZ™ varietyH7152 Roundup Ready™, HARTZ™ variety H7550 Roundup Ready™, HARTZ™variety H8001 Roundup Ready (HARTZ SEED, Stuttgart, Ark., U.S.A.);A0868, AGO901, A1553, A1900, AG1901, A1923, A2069, AG2101, AG2201,A2247, AG2301, A2304, A2396, AG2401, AG2501, A2506, A2553, AG2701,AG2702, A2704, A2833, A2869, AG2901, AG2902, AG3001, AG3002, A3204,A3237, A3244, AG3301, AG3302, A3404, A3469, AG3502, A3559, AG3601,AG3701, AG3704, AG3750, A3834, AG3901, A3904, A4045 AG4301, A4341,AG4401, AG4501, AG4601, AG4602, A4604, AG4702, AG4901, A4922, AG5401,A5547, AG5602, A5704, AG5801, AG5901, A5944, A5959, AG6101, QR4459 andQP4544 (Asgrow Seeds, Des Moines, Iowa, U.S.A.); DeKalb variety CX445(DeKalb, Ill.). An elite plant is any plant from an elite line.

B) Rhg4

The present invention provides a method for the production of a soybeanplant having an Rhg4 SCN resistant allele comprising: (A) crossing afirst soybean plant having an Rhg4 SCN resistant allele with a secondsoybean plant having an Rhg4 SCN sensitive allele to produce asegregating population; (B) screening the segregating population for amember having an Rhg4 SCN resistant allele with a first nucleic acidmolecule capable of specifically hybridizing to linkage group A2,wherein the first nucleic acid molecule specifically hybridizes to asecond nucleic acid molecule linked to the Rhg4 SCN resistant allele;and, (C) selecting the member for further crossing and selection.

Rhg4 is located on linkage group A2 (Matson and Williams, Crop Sci.5:447 (1965)). SCN resistant alleles of Rhg4 provide partial resistanceto SCN races 1 and 3 (U.S. Pat. No. 5,491,081). Together, rhg1 and Rhg4provide complete or nearly complete resistance to SCN race 3. Thedominant gene, Rhg4, was found to be closely linked to the seed coatcolor locus (i) (Matson and Williams, Crop Sci. 5:447 (1965)). The ilocus in Peking was also reported to be linked with a recessive gene forresistance to SCN (Sugiyama and Katsumi, Jpn. J. Breed. 16:83-86(1966)). It is possible that Rhg4 and the recessive gene linked to the ilocus are one and the same, which would call into question theclassification of Rhg4 as a dominant gene.

Using bioinformatic approaches the Rhg4 coding region is predicted tocontain 2 exons (coding coordinates 111805-113968 and 114684-115204 ofSEQ ID NO: 4). Rhg4 encodes an 894 amino acid polypeptide. Rhg4 codesfor a Xa21-like receptor kinase (SEQ ID NOs: 1099 and 1116-1119) (Songet al., Science 270, 1804-1806, (1995)). Rhg4 has an extracellular LRRdomain (Rhg4, SEQ ID NO: 1099, residues 34-44), a transmembrane domain(Rhg4 SEQ ID NO: 1099, residues 449-471), and STK domain (Rhg4, SEQ IDNO: 1099, residues 531-830). In a preferred embodiment, the LRR domainhas multiple LRR repeats. In a more preferred embodiment, the LRR domainhas 12 LRR repeats.

To identify proteins similar to the Rhg4 candidate, database searchesare performed using the predicted peptide sequences. The Rhg4 candidateshows similarity to TMK (Y07748)(73.0% similarity and 54.8% identity(CLUSTALW (default parameters))) and TMK1 PRECURSOR (70.6% similarityand 55.1% identity (CLUSTALW (default parameters))), which are rice andArabidopsis receptor kinases, respectively. The predicted LRRextracellular domain reveals similarity to TMK (Y07748)(70.1% similarityand 46.6% identity (CLUSTALW (default parameters))), TMK1 PRECURSOR(g1707642) (65.8% similarity and 48.8% identity (CLUSTALW (defaultparameters))), and F21J9.1 (g2213607) (65.5% similarity and 45.6%identity (CLUSTALW (default parameters))).

FIG. 2 is an alignment of the LRR domain of the Rhg4 gene. A consensussequence is shown as the top row. Each row of amino acids represents anLRR domain. The boxed region indicates the putative β-turn/β-sheetstructural motif postulated to be involved in ligand binding (Jones andJones, Adv. Bot. Res. Incorp. Adv. Plant Path. 24; 89-167 (1997)). Thehydrophobic leucine residues are thought to project into the core of theprotein while the flanking amino acids are thought to be solvent exposedwhere they may interact with the ligand (Kobe and Deisenhofer, Nature374; 183-186 (1995)). An “x” represents an arbitrary amino acid while an“a” represents a hydrophobic residue (leucine, isoleucine, methionine,valine, or phenylalanine). Amino acid substitutions between resistantand sensitive phenotypes are bordered by a double line. The amino acidsubstitution within the 35-57 region is a histidine/glutaminesubstitution, and the amino acid substitution within the 81-104 regionis a leucine/phenylalanine substitution.

As used herein, a naturally-occurring Rhg4 allele is any allele thatencodes for a protein having an extracellular LRR domain, atransmembrane domain, and STK domain where the naturally occurringallele is present on linkage group A2 and where certain Rgh4 alleles,but not all Rgh4 alleles, are capable of providing or contributing toresistance or partial resistance to a race of SCN. It is understood thatsuch an allele can, using, for example methods disclosed herein, bemanipulated so that the nucleic acid molecule encoding the protein is nolonger present on linkage group A2. It is also understood that such anallele can, using, for example methods disclosed herein, be manipulatedso that the nucleic acid molecule sequence is altered.

As used herein, an Rhg4 SCN resistant allele is any Rhg4 allele wherethat allele alone or in combination with other SCN resistant allelespresent in the plant, such as an rhg1 SCN resistant allele, providesresistance to a race of SCN, and that resistance is due, at least inpart, to the genetic contribution of the Rhg4 allele.

Any soybean plant having an Rhg4 SCN resistant allele can be used inconjunction with the present invention. Soybeans with known Rhg4 SCNresistant alleles can be used. Such soybeans include, but are notlimited to, PI548402 (Peking), PI437654 (Er-hej-jan), PI438489(Chiquita), PI507354 (Tokei 421), PI548655 (Forrest), PI548988(Pickett), PI88788, PI404198 (Sun Huan Do), PI404166 (Krasnoaarmejkaja),Hartwig, Manokin, Doles, Dyer, and Custer. In a preferred aspect, thesoybean plant having an Rhg4 SCN resistant allele is an Rhg4 haplotype 3allele in a plant having either an rhg1 haplotype 2 or rhg1 haplotype 4allele. Examples of soybeans with an Rhg4 haplotype 3 allele arePI548402 (Peking), PI88788, PI404198 (Sun huan do), PI438489 (Chiquita),PI437654 (Er-hej-jan), PI404166 (Krasnoaarmejkaja), PI548655 (Forrest),PI548988 (Pickett), and PI507354 (Tokei 421). In addition, using themethods or agents of the present invention, soybeans and wild relativesof soybeans such as Glycine soja can be screened for the presence ofRhg4 SCN resistant alleles.

Table 4 below is a table showing single nucleotide polymorphisms (SNPs)for three haplolotype sequences of Rhg4. TABLE 4 Identification Basenumber of contig 318O13_region_A3 Markers Hap PI number Line Ph Coat111933 112065 112101 112461 114066 scn279 scnb267 scn273 1 — A2069 Ryellow T A T A T 2 2 2 1 — A2869 R yellow T A T A T 2 2 2 1 — A3244 Syellow T A T A T 2 2 2 1 PI87631 Kindaizu R yellow T A T A T 2 2 2 1PI548389 Minsoy S yellow T A T A T 2 2 2 1 PI518664 Hutcheson S yellow TA T A T 2 2 2 1 PI548658 Lee 74 S yellow T A T A T — 2 2 2 PI540556 JackR yellow G A T A T 2 2 1 2 PI360843 Oshimashirome R yellow G A T A T — —— 2 PI423871 Toyosuzu R yellow G A T A T — — — 3 PI548402 Peking R blackG C C T G 1 1 1 3 PI88788 — R black G C C T G 1 1 1 3 PI404198 B (Sunhuan do) R black G C C T G 1 1 1 3 PI438489 B (Chiquita) R black G C C TG 1 1 1 3 PI437654 Er-hej-jan R black G C C T G 2 1 1 3 PI404166Krasnoaarmejkaja R black G C C T G 1 1 — 3 PI290136 Noir S black G C C TG 1 1 1 3 PI548655 Forrest R yellow G C C T G 1 1 1 3 PI548988 Pickett Ryellow G C C T G 1 1 1 3 PI507354 Tokei 421 R yellow G C C T G 1 1 1 N/API467312 Cha-mo-shi-dou R GnBr G C C T — 1 1 1 N/A PI209332 No. 4 Rblack T A T — — 2 2 2 N/A PI518672 Will S yellow T A T — T 2 2 2 N/API548667 Essex S yellow T A T — T 2 2 2

In Table 4, discrete haplotypes are designated 1 through 3. N/A refersto a haplotype that is not characterized. In Table 4, the PlantIntroduction classification number is indicated in the “PI#” column. Adash indicates that no PI number is known or assigned for the line underinvestigation. The line from which the sequences are derived isindicated in the “line” column, with a dash indicating an unknown orunnamed line. The “Ph.” column of Table 4 indicates whether a given linehas been reported to be resistant (R) to at least one race of SCN, orsensitive (S). The “coat” column shows the phenotypic coat color of aseed as either yellow, black, green/brown (GnBr), or unknown/unassigned(dash). At the I locus, black seeded varieties harbor the i allele forblack or imperfect black seed coat. In a preferred embodiment, the seedhas a yellow coat.

The nucleotide base located at each of 5 positions in each of thehaplotype sequences is shown in the columns labeled “Base number ofcontig 318O13_region_A3.” The base number at the top of each columncorrespond to the base number in the contig 318O13_region_A3 ofreference line A3244 (SEQ ID NO: 4). The letters G, A, C, and Tcorrespond to the bases guanine, adenine, cytosine, and thymine. A dashindicates that the identity of the base is unknown.

Three different simple sequence repeat (SSR) or microsatellite markersthat occur within the sequences, scn279 (SEQ ID NO: 292), scn267 (SEQ IDNO: 282), and scn273 (SEQ ID NO: 294), are listed under “markers.” Theallele of each marker occurring in a haplotype is indicated by a 1 or a2, with a dash indicating that the information is not determined.

Any soybean plant having an Rhg4 SCN sensitive allele can be used inconjunction with the present invention. Such soybeans include A3244,Will, Noir, Lee 74, Essex, Minsoy, A2704, A2833, AG3001, Williams,DK23-51, and Hutcheson. In a preferred aspect, the soybean plant havingan Rhg4 SCN sensitive allele is an Rhg4 A3244 allele. In addition, usingthe methods or agents of the present invention, soybeans and wildrelative of soybean such as Glycine soja can be screened for thepresence of Rhg4 SCN sensitive alleles.

In a preferred aspect, the source of either an Rhg4 SCN sensitive alleleor an Rhg4 SCN resistant allele, or more preferably both, is an eliteplant.

In table 5, below, rhg1 and Rhg4 haplotypes for various cultivars arecompared. TABLE 5 Identification Haplotype PI# Line Coat Ph. rhg4 rhg1 —A3244 yellow S 1 1 PI548402 Peking black R 3 2 PI404198 B (Sun huan do)black R 3 2 PI438489 B (Chiquita) black R 3 2 PI437654 Er-hej-jan blackR 3 2 PI404166 Krasnoaarmejkaja black R 3 2 PI548655 Forrest yellow R 32 PI548988 Pickett yellow R 3 2 PI507354 Tokei 421 yellow R 3 2 PI88788— black R 3 4 PI467312 Cha-mo-shi-dou GnBr R N/A 4 — Noir black S 3 6 —Jack yellow R 2 N/A PI360843 Oshimashirome yellow R 2 N/A PI423871Toyosuzu yellow R 2 3 PI209332 No. 4 black R N/A N/A PI87631 Kindaizuyellow R 1 — — Minsoy yellow S 1 N/A — Will yellow S N/A 4 — Hutchesonyellow S 1 6 — Lee 74 yellow S N/A 7 — Essex yellow S N/A N/A — A2069yellow R 1 N/A — A2869 yellow R 1 N/A

In Table 5, haplotypes, as used in Tables 2 through 4, are listed foreach line. N/A refers to a haplotype that is not characterized. ThePlant Introduction classification number is indicated in the “PI#”column. A dash indicates that no PI number is known or assigned for theline under investigation. The line from which the sequences are derivedis indicated in the “line” column, with a dash indicating an unknown orunnamed line. The “Ph.” column of table 5 indicates whether a given linehas been reported to be resistant (R) to at least one race of SCN, orsensitive (S). The “coat” column shows the phenotypic coat color of aseed as either yellow, black, green/brown (GnBr), or unknown/unassigned(dash). At the I locus, black seeded varieties harbor the i allele forblack or imperfect black seed coat. In a preferred embodiment, the seedhas a yellow coat.

Screening for rhg1 and Rhg4 Alleles

Any appropriate method can be used to screen for a plant having an rhg1SCN resistant allele. Any appropriate method can be used to screen for aplant having an Rhg4 SCN resistant allele. In a preferred aspect of thepresent invention, a nucleic acid marker of the present invention can beused (see section entitled “Screening for rhg1 and Rhg4 alleles” andsubsection (ii) of the section entitled “Agents”).

Additional markers, such as SSRs, AFLP markers, RFLP markers, RAPDmarkers, phenotypic markers, SNPs, isozyme markers, microarraytranscription profiles that are genetically linked to or correlated withalleles of a QTL of the present invention can be utilized (Walton, SeedWorld 22-29 (July, 1993); Burow and Blake, Molecular Dissection ofComplex Traits, 13-29, Eds. Paterson, CRC Press, New York (1988)).Methods to isolate such markers are known in the art. For example,locus-specific SSRs can be obtained by screening a genomic library forSSRs, sequencing of “positive” clones, designing primers which flank therepeats, and amplifying genomic DNA with these primers. The size of theresulting amplification products can vary by integral numbers of thebasic repeat unit. To detect a polymorphism, PCR products can beradiolabeled, separated on denaturing polyacrylamide gels, and detectedby autoradiography. Fragments with size differences >4 bp can also beresolved on agarose gels, thus avoiding radioactivity.

Other SSR markers may be utilized. Amplification of simple tandemrepeats, mainly of the [CA]_(n) type were reported by Litt and Luty,Amer. J. Human Genet. 44:397-401 (1989); Smeets et al., Human Genet.83:245-251 (1989); Tautz, Nucleic Acids Res. 17:6463-6472 (1989); Weberand May, Am. J. Hum. Genet. 44:388-396 (1989). Weber, Genomics 7:524-530(1990), reported that the level of polymorphism detected byPCR-amplified [CA]_(n) type SSRs depends on the number of the “perfect”(i.e., uninterrupted), tandemly repeated motifs. Below a certainthreshold (i.e., 12 CA-repeats), the SSRs were reported to be primarilymonomorphic. Above this threshold, however, the probability ofpolymorphism increases with SSR length. Consequently, long, perfectarrays of SSRs are preferred for the generation of markers, i.e., forthe design and synthesis of flanking primers.

Suitable primers can be deduced from DNA databases (e.g., Akkaya et al.,Genetics. 132:1131-1139 (1992)). Alternatively, size-selected genomiclibraries (200 to 500 bp) can be constructed by, for example, using thefollowing steps: (1) isolation of genomic DNA; (2) digestion with one ormore 4 base-specific restriction enzymes; (3) size-selection ofrestriction fragments by agarose gel electrophoresis, excision andpurification of the desire size fraction; (4) ligation of the DNA into asuitable vector and transformation into a suitable E. coli strain; (5)screening for the presence of SSRs by colony or plaque hybridizationwith a labeled probe; (6) isolation of positive clones and sequencing ofthe inserts; and (7) design of suitable primers flanking the SSR.

Establishing libraries with small, size-selected inserts can beadvantageous for SSR isolation for two reasons: (1) long SSRs are oftenunstable in E. coli, and (2) positive clones can be sequenced withoutsubcloning. A number of approaches have been reported for the enrichmentof SSRs in genomic libraries. Such enrichment procedures areparticularly useful if libraries are screened with comparatively raretri- and tetranucleotide repeat motifs. One such approach has beendescribed by Ostrander et al., Proc. Natl. Acad. Sci. (U.S.A).89:3419-3423 (1992), who reported the generation of a small-insertphagemid library in an E. coli strain deficient in UTPase (d8t) anduracil-N-glycosylase (ung) genes. In the absence of UTPase anduracil-N-glycosylase, dUTP can compete with dTTP for the incorporationinto DNA. Single-stranded phagemid DNA isolated from such a library canbe primed with [CA]_(n) and [TG]_(n) primers for second strandsynthesis, and the products used to transform a wild-type E. colistrain. Since under these conditions there will be selection againstsingle-stranded, uracil-containing DNA molecules, the resulting librarywill consist of primer-extended, double-stranded products and an about50-fold enrichment in CA-repeats.

Other reported enrichment strategies rely on hybridization selection ofsimple sequence repeats prior to cloning (Karagyozov et al., NucleicAcids Res. 21:3911-3912 (1993); Armour et al., Hum. Mol. Gen. 3:599-605(1994); Kijas et al., Genome 38:349-355 (1994); Kandpal et al., Proc.Natl. Acad. Sci. (U.S.A.) 91:88-92 (1994); Edwards et al., Am. J. Hum.Genet. 49:746-756 (1991)). Hybridization selection, can for example,involve the following steps: (1) genomic DNA is fragmented, either bysonication, or by digestion with a restriction enzyme; (2) genomic DNAfragments are ligated to adapters that allow a “whole genome PCR” atthis or a later stage of the procedure; (3) genomic DNA fragments areamplified, denatured and hybridized with single-stranded SSR sequencesbound to a nylon membrane; (4) after washing off unbound DNA,hybridizing fragments enriched for SSRs are eluted from the membrane byboiling or alkali treatment, reamplified using adapter-complementaryprimers, and digested with a restriction enzyme to remove the adapters;and (5) DNA fragments are ligated into a suitable 15 vector andtransformed into a suitable E. coli strain. SSRs can be found in up to50-70% of the clones obtained from these procedures (Armour et al., Hum.Mol. Gen. 3:599-605 (1994); Edwards et al., Am. J. Hum. Genet.49:746-756 (1991)).

An alternative hybridization selection strategy was reported by Kijas etal., Genome 38:599-605 (1994), which replaced the nylon membrane withbiotinylated, SSR-complementary oligonucleotides attached tostreptavidin-coated magnetic particles. SSR-containing DNA fragments areselectively bound to the magnetic beads, reamplified,restriction-digested and cloned.

It is further understood that other additional markers on linkage groupG or A2 may be utilized (Morgante et al., Genome 37:763-769 (1994)). Asused herein, reference to the linkage group of G or A2 refers to thelinkage group that corresponds to linkage groups U5 and U3, respectivelyfrom the genetic map of Glycine max (Mansur et al., Crop Sci. 36:1327-1336 (1996), and linkage groups G and A2, respectively, of Glycinemax x. Glycine soja (Shoemaker et al., Genetics 144: 329-336 (1996))that is present in Glycine soja (Soybase, an Agricultural ResearchService, United States Department of Agriculture (http-129.186.26.940and USDA-Agricultural Research Service: http-www.ars.usda.gov/)).

PCR-amplified SSRs can be used, because they are locus-specific,codominant, occur in large numbers and allow the unambiguousidentification of alleles. Standard PCR-amplified SSR protocols useradioisotopes and denaturing polyacrylamide gels to detect amplifiedSSRs. In many situations, however, allele sizes are sufficientlydifferent to be resolved on high percentage agarose gels in combinationwith ethidium bromide staining (Bell and Ecker, Genomics 19:137-144(1994); Becker and Heun, Genome 38:991-998 (1995); Huttel, Ph.D. Thesis,University of Frankfurt, Germany (1996)). High resolution withoutapplying radioactivity is also provided by nondenaturing polyacrylamidegels in combination with either ethidium bromide (Scrimshaw,Biotechniques 13:2189 (1992)) or silver straining (Klinkicht and Tautz,Molecular Ecology 1: 133-134 (1992); Neilan et al., Biotechniques17:708-712 (1994)). An alternative of PCR-amplified SSRs typing involvesthe use of fluorescent primers in combination with a semi-automated DNAsequencer (Schwengel et al., Genomics 22:46-54 (1994)). Fluorescent PCRproducts can be detected by real-time laser scanning during gelelectrophoresis. An advantage of this technology is that differentamplification reactions as well as a size marker (each labeled with adifferent fluorophore) can be combined into one lane duringelectrophoresis. Multiplex analysis of up to 24 different SSR loci perlane has been reported (Schwengel et al., Genomics 22:46-54 (1994)).

The detection of polymorphic sites in a sample of DNA may be facilitatedthrough the use of nucleic acid amplification methods. Such methodsspecifically increase the concentration of polynucleotides that span thepolymorphic site, or include that site and sequences located eitherdistal or proximal to it. Such amplified molecules can be readilydetected by gel electrophoresis or other means.

The most preferred method of achieving such amplification employs thepolymerase chain reaction (“PCR”) (Mullis et al., Cold Spring HarborSymp. Quant. Biol. 51:263-273 (1986); Erlich et al., European PatentAppln. 50,424; European Patent Appln. 84,796, European PatentApplication 258,017, European Patent Appln. 237,362; Mullis, EuropeanPatent Appln. 201,184; Mullis et al., U.S. Pat. No. 4,683,202; Erlich,U.S. Pat. No. 4,582,788; and Saiki et al., U.S. Pat. No. 4,683,194),using primer pairs that are capable of hybridizing to the proximalsequences that define a polymorphism in its double-stranded form.

In lieu of PCR, alternative methods, such as the “Ligase Chain Reaction”(“LCR”) may be used (Barany, Proc. Natl. Acad. Sci. (U.S.A.) 88:189-193(1991)). LCR uses two pairs of oligonucleotide probes to exponentiallyamplify a specific target. The sequences of each pair ofoligonucleotides is selected to permit the pair to hybridize to abuttingsequences of the same strand of the target. Such hybridization forms asubstrate for a template-dependent ligase. As with PCR, the resultingproducts thus serve as a template in subsequent cycles and anexponential amplification of the desired sequence is obtained.

LCR can be performed with oligonucleotides having the proximal anddistal sequences of the same strand of a polymorphic site. In oneembodiment, either oligonucleotide will be HE designed to include theactual polymorphic site of the polymorphism. In such an embodiment, thereaction conditions are selected such that the oligonucleotides can beligated together only if the target molecule either contains or lacksthe specific nucleotide that is complementary to the polymorphic sitepresent on the oligonucleotide. Alternatively, the oligonucleotides maybe selected such that they do not include the polymorphic site (see,Segev, PCT Application WO 90/01069).

The “Oligonucleotide Ligation Assay” (“OLA”) may alternatively beemployed (Landegren et al., Science 241:1077-1080 (1988)). The OLAprotocol uses two oligonucleotides that are designed to be capable ofhybridizing to abutting sequences of a single strand of a target. OLA,like LCR, is particularly suited for the detection of point mutations.Unlike LCR, however, OLA results in “linear” rather than exponentialamplification of the target sequence.

Nickerson et al. have described a nucleic acid detection assay thatcombines attributes of PCR and OLA (Nickerson et al., Proc. Natl. Acad.Sci. (U.S.A.) 87:8923-8927 (1990)). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA. In addition to requiring multiple, and separate,processing steps, one problem associated with such combinations is thatthey inherit all of the problems associated with PCR and OLA.

Schemes based on ligation of two (or more) oligonucleotides in thepresence of a nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the di-oligonucleotide, arealso known (Wu et al., Genomics 4:560-569 (1989)), and may be readilyadapted to the purposes of the present invention.

Other known nucleic acid amplification procedures, such asallele-specific oligomers, branched DNA technology, transcription-basedamplification systems, or isothermal amplification methods may also beused to amplify and analyze such polymorphisms (Malek et al., U.S. Pat.No. 5,130,238; Davey et al., European Patent Application 329,822;Schuster et al., U.S. Pat. No. 5,169,766; Miller et al., PCT PatentApplication WO 89/06700; Kwoh, et al., Proc. Natl. Acad. Sci. (U.S.A.)86:1173-1177 (1989); Gingeras et al., PCT Patent Application WO88/10315; Walker et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396(1992)).

Polymorphisms can also be identified by Single Strand ConformationPolymorphism (SSCP) analysis. SSCP is a method capable of identifyingmost sequence variations in a single strand of DNA, typically between150 and 250 nucleotides in length (Elles, Methods in Molecular Medicine:Molecular Diagnosis of Genetic Diseases, Humana Press (1996); Orita etal., Genomics 5: 874-879 (1989)). Under denaturing conditions a singlestrand of DNA will adopt a conformation that is uniquely dependent onits sequence conformation. This conformation usually will be different,even if only a single base is changed. Most conformations have beenreported to alter the physical configuration or size sufficiently to bedetectable by electrophoresis. A number of protocols have been describedfor SSCP including, but not limited to, Lee et al., Anal. Biochem. 205:289-293 (1992); Suzuki et al., Anal. Biochem. 192: 82-84 (1991); Lo etal., Nucleic Acids Research 20: 1005-1009 (1992); Sarkar et al.,Genomics 13:441-443 (1992). It is understood that one or more of thenucleic acids of the present invention can be utilized as markers orprobes to detect polymorphisms by SSCP analysis.

Polymorphisms may also be found using random amplified polymorphic DNA(RAPD) (Williams et al., Nucl. Acids Res. 18: 6531-6535 (1990)) andcleaveable amplified polymorphic sequences (CAPS) (Lyamichev et al.,Science 260: 778-783 (1993)). It is understood that one or more of thenucleic acid molecules of the present invention can be utilized asmarkers or probes to detect polymorphisms by RAPD or CAPS analysis.

The identification of a polymorphism can be determined in a variety ofways. By correlating the presence or absence of it in a plant with thepresence or absence of a phenotype, it is possible to predict thephenotype of that plant. If a polymorphism creates or destroys arestriction endonuclease cleavage site, or if it results in the loss orinsertion of DNA (e.g., a variable nucleotide tandem repeat (VNTR)polymorphism), it will alter the size or profile of the DNA fragmentsthat are generated by digestion with that restriction endonuclease. Assuch, individuals that possess a variant sequence can be distinguishedfrom those having the original sequence by restriction fragmentanalysis. Polymorphisms that can be identified in this manner are termed“restriction fragment length polymorphisms” (“RFLPs”). RFLPs have beenwidely used in human and plant genetic analyses (Glassberg, UK PatentApplication 2135774; Skolnick et al., Cytogen. Cell Genet. 32:58-67(1982); Botstein et al., Ann. J. Hum. Genet. 32:314-331 (1980); Fischeret al. (PCT Application WO90/13668); Uhlen, PCT ApplicationWO90/11369)).

A central attribute of “single nucleotide polymorphisms,” or “SNPs” isthat the site of the polymorphism is at a single nucleotide. SNPs havecertain reported advantages over RFLPs and VNTRs. First, SNPs are morestable than other classes of polymorphisms. Their spontaneous mutationrate is approximately 10⁻⁹ (Komberg, DNA Replication, W.H. Freeman &Co., San Francisco, 1980), approximately 1,000 times less frequent thanVNTRs (U.S. Pat. No. 5,679,524). Second, SNPs occur at greaterfrequency, and with greater uniformity than RFLPs and VNTRs. As SNPsresult from sequence variation, new polymorphisms can be identified bysequencing random genomic or cDNA molecules. SNPs can also result fromdeletions, point mutations and insertions. Any single base alteration,whatever the cause, can be an SNP. The greater frequency of SNPs meansthat they can be more readily identified than the other classes ofpolymorphisms.

SNPs and insertion/deletions can be detected by methods, by any of avariety of methods including those disclosed in U.S. Pat. Nos.5,210,015; 5,876,930 and 6,030,787 in which an oligonucleotide probehaving reporter and quencher molecules is hybridized to a targetpolynucleotide. The probe is degraded by 5′→3′ exonuclease activity of anucleic acid polymerase. A useful assay is available from AB Biosystems(850 Lincoln Centre Drive, Foster City, Calif.) as the Taqman® assay.

Specific nucleotide variations such as SNPs and insertion/deletions canalso be detected by labeled base extension methods as disclosed in U.S.Pat. Nos. 6,004,744; 6,013,431; 5,595,890; 5,762,876; and 5,945,283.These methods are based on primer extension and incorporation ofdetectable nucleoside triphosphates. The primer is designed to anneal tothe sequence immediately adjacent to the variable nucleotide which canbe can be detected after incorporation of as few as one labelednucleoside triphosphate. U.S. Pat. No. 5,468,613 discloses allelespecific oligonucleotide hybridizations where single or multiplenucleotide variations in nucleic acid sequence can be detected innucleic acids by a process in which the sequence containing thenucleotide variation is amplified, spotted on a membrane and treatedwith a labeled sequence-specific oligonucleotide probe.

Such methods also include the direct or indirect sequencing of the site,the use of restriction enzymes where the respective alleles of the sitecreate or destroy a restriction site, the use of allele-specifichybridization probes, the use of antibodies that are specific for theproteins encoded by the different alleles of the polymorphism or byother biochemical interpretation. SNPs can be sequenced by a number ofmethods. Two basic methods may be used for DNA sequencing, the chaintermination method of Sanger et al., Proc. Natl. Acad. Sci. (U.S.A.) 74:5463-5467 (1977), and the chemical degradation method of Maxam andGilbert, Proc. Nat. Acad. Sci. (U.S.A.) 74: 560-564 (1977). Automationand advances in technology such as the replacement of radioisotopes withfluorescence-based sequencing have reduced the effort required tosequence DNA (Craxton, Methods, 2: 20-26 (1991); Ju et al., Proc. Natl.Acad. Sci. (U.S.A.) 92: 4347-4351 (1995); Tabor and Richardson, Proc.Natl. Acad. Sci. (U.S.A.) 92: 6339-6343 (1995)). Automated sequencersare available from, for example, Pharmacia Biotech, Inc., Piscataway,N.J. (Pharmacia ALF), LI-COR, Inc., Lincoln, Nebr. (LI-COR 4,000) andMillipore, Bedford, Mass. (Millipore BaseStation).

In addition, advances in capillary gel electrophoresis have also reducedthe effort required to sequence DNA and such advances provide a rapidhigh resolution approach for sequencing DNA samples (Swerdlow andGesteland, Nucleic Acids Res. 18:1415-1419 (1990); Smith, Nature349:812-813 (1991); Luckey et al., Methods Enzymol. 218:154-172 (1993);Lu et al., J. Chromatog. A. 680:497-501 (1994); Carson et al., Anal.Chem. 65:3219-3226 (1993); Huang et al., Anal. Chem. 64:2149-2154(1992); Kheterpal et al., Electrophoresis 17:1852-1859 (1996); Quesadaand Zhang, Electrophoresis 17:1841-1851 (1996); Baba, Yakugaku Zasshi117:265-281 (1997), Marino, Appl. Theor. Electrophor. 5:1-5 (1995)).

The genetic linkage of marker molecules can be established by a genemapping model such as, without limitation, the flanking marker modelreported by Lander and Botstein, Genetics, 121:185-199 (1989), and theinterval mapping, based on maximum likelihood methods described byLander and Botstein, Genetics, 121:185-199 (1989), and implemented inthe software package MAPMAKER/QTL (Lincoln and Lander, Mapping GenesControlling Quantitative Traits Using MAPMAKER/QTL, Whitehead Institutefor Biomedical Research, Massachusetts, (1990). Additional softwareincludes Qgene, Version 2.23 (1996), Department of Plant Breeding andBiometry, 266 Emerson Hall, Cornell University, Ithaca, N.Y.). Use ofQgene software is a particularly preferred approach.

A maximum likelihood estimate (MLE) for the presence of a marker iscalculated, together with an MLE assuming no QTL effect, to avoid falsepositives. A log₁₀ of an odds ratio (LOD) is then calculated as:LOD=log₁₀ (MLE for the presence of a QTL(MLE given no linked QTL).

The LOD score essentially indicates how much more likely the data are tohave arisen assuming the presence of a QTL than in its absence. The LODthreshold value for avoiding a false positive with a given confidence,say 95%, depends on the number of markers and the length of the genome.Graphs indicating LOD thresholds are set forth in Lander and Botstein,Genetics, 121:185-199 (1989), and further described by Arús andMoreno-González, Plant Breeding, Hayward, Bosemark, Romagosa (eds.)Chapman & Hall, London, pp. 314-331 (1993).

Additional models can be used. Many modifications and alternativeapproaches to interval mapping have been reported, including the use ofnon-parametric methods (Kruglyak and Lander, Genetics, 139:1421-1428(1995)). Multiple regression methods or models can be also be used, inwhich the trait is regressed on a large number of markers (Jansen,Biometrics in Plant Breed, van Oijen, Jansen (eds.) Proceedings of theNinth Meeting of the Eucarpia Section Biometrics in Plant Breeding, TheNetherlands, pp. 116-124 (1994); Weber and Wricke, Advances in PlantBreeding, Blackwell, Berlin, 16 (1994)). Procedures combining intervalmapping with regression analysis, whereby the phenotype is regressedonto a single putative QTL at a given marker interval, and at the sametime onto a number of markers that serve as ‘cofactors,’ have beenreported by Jansen and Stam, Genetics, 136:1447-1455 (1994) and Zeng,Genetics, 136:1457-1468 (1994). Generally, the use of cofactors reducesthe bias and sampling error of the estimated QTL positions (Utz andMelchinger, Biometrics in Plant Breeding, van Oijen, Jansen (eds.)Proceedings of the Ninth Meeting of the Eucarpia Section Biometrics inPlant Breeding, The Netherlands, pp. 195-204 (1994), thereby improvingthe precision and efficiency of QTL mapping (Zeng, Genetics,136:1457-1468 (1994)). These models can be extended to multi-environmentexperiments to analyze genotype-environment interactions (Jansen et al.,Theo. Appl. Genet. 91:33-37 (1995)).

Selection of an appropriate mapping or segregation populations isimportant to map construction. The choice of appropriate mappingpopulation depends on the type of marker systems employed (Tanksley etal., Molecular mapping plant chromosomes. Chromosome structure andfunction: Impact of new concepts J. P. Gustafson and R. Appels (eds.),Plenum Press, New York, pp. 157-173 (1988)). Consideration must be givento the source of parents (adapted vs. exotic) used in the mappingpopulation. Chromosome pairing and recombination rates can be severelydisturbed (suppressed) in wide crosses (adapted x exotic) and generallyyield greatly reduced linkage distances. Wide crosses will usuallyprovide segregating populations with a relatively large array ofpolymorphisms when compared to progeny in a narrow cross (adapted xadapted).

As used herein, the progeny include not only, without limitation, theproducts of any cross (be it a backcross or otherwise) between twoplants, but all progeny whose pedigree traces back to the originalcross. Specifically, without limitation, such progeny include plantsthat have 12.5% or less genetic material derived from one of the twooriginally crossed plants. As used herein, a second plant is derivedfrom a first plant if the second plant's pedigree includes the firstplant.

An F₂ population is the first generation of selfing after the hybridseed is produced. Usually a single F₁ plant is selfed to generate apopulation segregating for all the genes in Mendelian (1:2:1) fashion.Maximum genetic information is obtained from a completely classified F₂population using a codominant marker system (Mather, Measurement ofLinkage in Heredity: Methuen and Co., (1938)). In the case of dominantmarkers, progeny tests (e.g., F₃, BCF₂) are required to identify theheterozygotes, thus making it equivalent to a completely classified F₂population. However, this procedure is often prohibitive because of thecost and time involved in progeny testing. Progeny testing of F₂individuals is often used in map construction where phenotypes do notconsistently reflect genotype (e.g., disease resistance) or where traitexpression is controlled by a QTL. Segregation data from progeny testpopulations (e.g., F₃ or BCF₂) can be used in map construction.Marker-assisted selection can then be applied to cross progeny based onmarker-trait map associations (F₂, F₃), where linkage groups have notbeen completely disassociated by recombination events (i.e., maximumdisequilibrium).

Recombinant inbred lines (RIL) (genetically related lines; usually >F₅,developed from continuously selfing F₂ lines towards homozygosity) canbe used as a mapping population. Information obtained from dominantmarkers can be maximized by using RIL because all loci are homozygous ornearly so. Under conditions of tight linkage (i.e., about <10%recombination), dominant and co-dominant markers evaluated in RILpopulations provide more information per individual than either markertype in backcross populations (Reiter et al., Proc. Natl. Acad. Sci.(U.S.A.) 89:1477-1481 (1992)). However, as the distance between markersbecomes larger (i.e., loci become more independent), the information inRIL populations decreases dramatically when compared to codominantmarkers.

Backcross populations (e.g., generated from a cross between a successfulvariety (recurrent parent) and another variety (donor parent) carrying atrait not present in the former) can be utilized as a mappingpopulation. A series of backcrosses to the recurrent parent can be madeto recover most of its desirable traits. Thus a population is createdconsisting of individuals nearly like the recurrent parent but eachindividual carries varying amounts or mosaic of genomic regions from thedonor parent. Backcross populations can be useful for mapping dominantmarkers if all loci in the recurrent parent are homozygous and the donorand recurrent parent have contrasting polymorphic marker alleles (Reiteret al., Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481 (1992)).Information obtained from backcross populations using either codominantor dominant markers is less than that obtained from F₂ populationsbecause one, rather than two, recombinant gametes are sampled per plant.Backcross populations, however, are more informative (at low markersaturation) when compared to RELs as the distance between linked lociincreases in RIL populations (i.e., about 0.15% recombination).Increased recombination can be beneficial for resolution of tightlinkages, but may be undesirable in the construction of maps with lowmarker saturation.

Near-isogenic lines (NIL) created by many backcrosses to produce anarray of individuals that are nearly identical in genetic compositionexcept for the trait or genomic region under interrogation can be usedas a mapping population. In mapping with NILs, only a portion of thepolymorphic loci are expected to map to a selected region.

Bulk segregant analysis (BSA) is a method developed for the rapididentification of linkage between markers and traits of interest(Michelmore, et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:9828-9832(1991)). In BSA, two bulked DNA samples are drawn from a segregatingpopulation originating from a single cross. These bulks containindividuals that are identical for a particular trait (resistant orsensitive to particular disease) or genomic region but arbitrary atunlinked regions (i.e., heterozygous). Regions unlinked to the targetregion will not differ between the bulked samples of many individuals inBSA.

Plants generated using a method of the present invention can be part ofor generated from a breeding program. The choice of breeding methoddepends on the mode of plant reproduction, the heritability of thetrait(s) being improved, and the type of cultivar used commercially(e.g., F₁ hybrid cultivar, pureline cultivar, etc). Selected,non-limiting approaches, for breeding the plants of the presentinvention are set forth below. A breeding program can be enhanced usingmarker assisted selection of the progeny of any cross. It is furtherunderstood that any commercial and non-commercial cultivars can beutilized in a breeding program. Factors such as, for example, emergencevigor, vegetative vigor, stress tolerance, disease resistance,branching, flowering, seed set, seed size, seed density, standability,and threshability etc. will generally dictate the choice.

For highly heritable traits, a choice of superior individual plantsevaluated at a single location will be effective, whereas for traitswith low heritability, selection should be based on mean values obtainedfrom replicated evaluations of families of related plants. Popularselection methods commonly include pedigree selection, modified pedigreeselection, mass selection, and recurrent selection. In a preferredembodiment a backcross or recurrent breeding program is undertaken.

The complexity of inheritance influences choice of the breeding method.Backcross breeding can be used to transfer one or a few favorable genesfor a highly heritable trait into a desirable cultivar. This approachhas been used extensively for breeding disease-resistant cultivars.Various recurrent selection techniques are used to improvequantitatively inherited traits controlled by numerous genes. The use ofrecurrent selection in self-pollinating crops depends on the ease ofpollination, the frequency of successful hybrids from each pollination,and the number of hybrid offspring from each successful cross.

Breeding lines can be tested and compared to appropriate standards inenvironments representative of the commercial target area(s) for two ormore generations. The best lines are candidates for new commercialcultivars; those still deficient in traits may be used as parents toproduce new populations for further selection.

One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations can provide a better estimate of its genetic worth. Abreeder can select and cross two or more parental lines, followed byrepeated selfing and selection, producing many new genetic combinations.

The development of new soybean cultivars requires the development andselection of soybean varieties, the crossing of these varieties andselection of superior hybrid crosses. The hybrid seed can be produced bymanual crosses between selected male-fertile parents or by using malesterility systems. Hybrids are selected for certain single gene traitssuch as pod color, flower color, seed yield, pubescence color orherbicide resistance which indicate that the seed is truly a hybrid.Additional data on parental lines, as well as the phenotype of thehybrid, influence the breeder's decision whether to continue with thespecific hybrid cross.

Pedigree breeding and recurrent selection breeding methods can be usedto develop cultivars from breeding populations. Breeding programscombine desirable traits from two or more cultivars or variousbroad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. New cultivarscan be evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops. Two parents who possess favorable, complementarytraits are crossed to produce an F₁. An F₂ population is produced byselfing one or several F₁'s. Selection of the best individuals in thebest families is performed. Replicated testing of families can begin inthe F₄ generation to improve the effectiveness of selection for traitswith low heritability. At an advanced stage of inbreeding (i.e., F₆ andF₇), the best lines or mixtures of phenotypically similar lines aretested for potential release as new cultivars.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line, which is the recurrent parent. The source of the traitto be transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting parent is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, soybean breeders commonly harvest one ormore pods from each plant in a population and thresh them together toform a bulk. Part of the bulk is used to plant the next generation andpart is put in reserve. The procedure has been referred to as modifiedsingle-seed descent or the pod-bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster to thresh pods with a machine than to remove oneseed from each by hand for the single-seed procedure. The multiple-seedprocedure also makes it possible to plant the same number of seeds of apopulation each generation of inbreeding.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Fehr, Principles of Cultivar Development Vol. 1, pp. 2-3(1987)).

In a preferred aspect of the present invention the source of the rhg1SCN resistant allele for use in a breeding program is derived from aplant selected from the group consisting of PI548402 (Peking), PI200499,A2869, Jack, A2069, PI209332 (No:4), PI404166 (Krasnoaarmejkaja),PI404198 (Sun huan do), PI437654 (Er-hej-jan), PI438489 (Chiquita),PI507354 (Tokei 421), PI548655 (Forrest), PI548988 (Pickett), PI84751,PI437654, PI40792, Pyramid, Nathan, AG2201, A3469, AG3901, A3904,AG4301, AG4401, AG4501, AG4601, PION9492, PI88788, Dyer, Custer,Manokin, Doles, and SCN resistant progeny thereof (USDA, SoybeanGermplasm Collection, University of Illinois, Illinois). In a morepreferred aspect, the source of the rhg1 SCN resistant allele for use ina breeding program is derived from a plant selected from the groupconsisting of PI548402 (Peking), PI404166 (Krasnoaarmejkaja), PI404198(Sun huan do), PI437654 (Er-hej-jan), PI438489 (Chiquita), PI507354(Tokei 421), PI548655 (Forrest), PI548988 (Pickett), PI84751, PI437654,PI40792, and SCN resistant progeny thereof.

In a preferred aspect of the present invention the source of the rhg1SCN sensitive allele for use in a breeding program is derived from aplant selected from the group consisting of A3244, A2833, AG3001,Williams, Will, A2704, Noir, DK23-5 1, Lee 74, Essex, Minsoy, A1923,Hutcheson, and SCN sensitive progeny thereof. In a more preferredaspect, the source of the rhg1 SCN sensitive allele for use in abreeding program is derived from an A3244 plant, and SCN sensitiveprogeny thereof.

In a preferred aspect of the present invention the source of the Rhg4SCN resistant allele for use in a breeding program is derived from aplant selected from the group consisting of PI548402 (Peking), PI437654(Er-hejjan), PI438489 (Chiquita), PI507354 (Tokei 421), PI548655(Forrest), PI548988 (Pickett), PI88788, PI404198 (Sun Huan Do), PI404166(Krasnoaarmejkaja), Hartwig, Manokin, Doles, Dyer, Custer, and SCNresistant progeny thereof. In a more preferred aspect, the source of theRhg4 SCN resistant allele for use in a breeding program is derived froma plant selected from the group consisting of PI548402 (Peking),PI88788, PI404198 (Sun huan do), PI438489 (Chiquita), PI437654(Er-hej-jan), PI404166 (Krasnoaarmejkaja), PI548655 (Forrest), PI548988(Pickett), PI507354 (Tokei 421), and SCN resistant progeny thereof.

In a preferred aspect of the present invention the source of the Rhg4SCN sensitive allele for use in a breeding program is derived from aplant selected from the group consisting of A3244, Will, Noir, Lee 74,Essex, Minsoy, A2704, A2833, AG3001, Williams, DK23-51, and Hutcheson,and SCN sensitive progeny thereof. In a more preferred aspect, thesource of the Rhg4 SCN sensitive allele for use in a breeding program isderived from an A3244 plant, and SCN sensitive progeny thereof.

As used herein linkage of a nucleic acid sequence with another nucleicacid sequence may be genetic or physical. In a preferred embodiment, anucleic acid marker is genetically linked to either rhg1 or Rhg4, wherethe marker nucleic acid molecule exhibits a LOD score of greater than2.0, as judged by interval mapping, for SCN resistance or partialresistance, preferably where the marker nucleic acid molecule exhibits aLOD score of greater than 3.0, as judged by interval mapping, for SCNresistance or partial resistance, more preferably where the markernucleic acid molecule exhibits a LOD score of greater than 3.5, asjudged by interval mapping, for SCN resistance or partial resistance andeven more preferably where the marker nucleic acid molecule exhibits aLOD score of about 4.0, as judged by interval mapping, for SCNresistance or partial resistance based on maximum likelihood methodsdescribed by Lander and Botstein, Genetics, 121:185-199 (1989), andimplemented in the software package MAPMAKER/QTL (defaultparameters)(Lincoln and Lander, Mapping Genes Controlling QuantitativeTraits Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research,Massachusetts, (1990)).

In another embodiment the nucleic acid molecule may be physically linkedto either rhg1 or Rhg4. In a preferred embodiment, the nucleic acidmarker specifically hybridizes to a nucleic acid molecule having asequence that is present on linkage group G within 500 kb or 100 kb,more preferably within 50 kb, even more preferably within 25 kb of anrhg1 allele, where the rgh1 allele is preferably a sensitive allele, andmore preferably a sensitive allele from A3244. In a preferred embodimentthe nucleic acid marker is capable of specifically hybridizing to anucleic acid molecule having a sequence that is present on linkage groupA2 within 500 kb or 100 kb, more preferably within 50 kb, even morepreferably within 25 kb of an Rhg4 allele, where the Rgh4 allele ispreferably a sensitive allele, and more preferably a sensitive allelefrom A3244.

The present invention provides a method of investigating an rhg1haplotype of a soybean plant comprising: (A) isolating nucleic acidmolecules from the soybean plant; (B) determining the nucleic acidsequence of an rhg1 allele or part thereof; and, (C) comparing thenucleic acid sequence of the rhg1 allele or part thereof to a referencenucleic acid sequence.

As used herein, the term “investigating” refers to any method capable ofdetecting a feature, such as a polymorphism or haplotype. Nucleic acidmolecules only need to be isolated from a soybean plant to the degree ofpurity necessary for the task required or to a greater purity ifdesired. A person of ordinary skill in the art has available techniquesto isolate nucleic acid molecules from plants to a sufficient purity,for example without limitation, to sequence the desired region of thenucleic acid molecule or to carry out a marker assay.

The determination of an rhg1 or Rhg4 allele or part thereof may becarried out using any technique. Illustration of such techniques includetechniques that provide the nucleic acid sequence for an rhg1 or rhg4allele or part thereof include amplification of a desired allele or partthereof (see, for example, the Examples and SEQ ID NOs: 8-53). In apreferred embodiment, the nucleic acid sequence determined is that of anexon of an rhg1 allele, more preferably exon 1 or exon 3 of an rhg1allele, or of an LRR domain. In another preferred embodiment, a singlenucleotide is determined. In another preferred embodiment, the nucleicacid sequence determined is that of an LRR domain.

A comparison of a sequence with a reference sequence can be carried outwith any appropriate sequence comparison method.

As used herein, a reference sequence is any rhg1 allele sequence orconsensus sequence. A reference sequence may be a nucleic acid sequenceor an amino acid sequence. In a preferred embodiment, the referencesequence is any SCN resistant rhg1 allele sequence. In a furtherpreferred embodiment, the rhg1 reference sequence is selected from thegroup consisting of SEQ ID NOs: 2, 3, 5, 6, 8-23, 28-43, 1097, 1098, and1100-1115.

The present invention provides a method of investigating an Rhg4haplotype of a soybean plant comprising: (A) isolating nucleic acidmolecules from the soybean plant; (B) determining the nucleic acidsequence of an Rhg4 allele or part thereof; and (C) comparing thenucleic acid sequence of the Rhg4 allele or part thereof to a referencenucleic acid sequence.

As used herein, a reference sequence is any Rhg4 allele sequence orconsensus sequence. A reference sequence ma be a nucleic acid sequenceor an amino acid sequence. In a preferred embodiment, the referencesequence is any SCN resistant Rhg4 allele sequence. In a furtherpreferred embodiment, the Rhg4 reference sequence is selected from thegroup consisting of SEQ ID NOs: 4, 7, 44-47, 50-53, 1099, and 1116-1119.

The present invention provides a method of introgressing SCN resistanceor partial SCN resistance into a soybean plant comprising: performingmarker assisted selection of the soybean plant with a nucleic acidmarker, wherein the nucleic acid marker specifically hybridizes with anucleic acid molecule having a first nucleic acid sequence that isphysically linked to a second nucleic acid sequence that is located onlinkage group G of soybean A3244, wherein the second nucleic acidsequence is within 500 kb of a third nucleic acid sequence which iscapable of specifically hybridizing with the nucleic acid sequence ofSEQ ID NO: 5, 6, complements thereof, or fragments thereof; and,selecting the soybean plant based on the marker assisted selection.

The present invention provides a method of introgressing SCN resistanceor partial SCN resistance into a soybean plant comprising: performingmarker assisted selection of the soybean plant with a nucleic acidmarker, wherein the nucleic acid marker specifically hybridizes with anucleic acid molecule having a first nucleic acid sequence that isphysically linked to a second nucleic acid sequence that is located onlinkage group A2 of soybean A3244, wherein the second nucleic acidsequence is within 500 kb of a third nucleic acid sequence which iscapable of specifically hybridizing with the nucleic acid sequence ofSEQ ID NO: 7, complements thereor, or fragments thereof; and, selectingthe soybean plant based on the marker assisted selection. Markerassisted introgression of traits into plants has been reported. Markerassisted introgression involves the transfer of a chromosome regiondefined by one or more markers from one germplasm to a second germplasm.In a preferred embodiment the introgression is carried out bybackcrossing with an rhg1 or Rhg4 SCN resistant soybean recurrentparent.

In light of the current disclosure, plant introductions and germplasmcan be screened with a marker nucleic acid molecule of the presentinvention to screen for alleles of rhg1 or Rhg4 using one or more oftechniques disclosed herein or known in the art.

The present invention also provides for parts of the plants produced bya method of the present invention. Plant parts, without limitation,include seed, endosperm, ovule and pollen. In a particularly preferredembodiment of the present invention, the plant part is a seed.

Plants or parts thereof produced by a method of the present inventionmay be grown in culture and regenerated. Methods for the regeneration ofsoybean plants from various tissue types and methods for the tissueculture of soybean are known in the art (See, for example, Widholm etal., In Vitro Selection and Culture-induced Variation in Soybean, InSoybean: Genetics, Molecular Biology and Biotechnology, Eds. Verma andShoemaker, CAB International, Wallingford, Oxon, England (1996)).Regeneration techniques for plants such as soybean can use as thestarting material a variety of tissue or cell types. With soybean inparticular, regeneration processes have been developed that begin withcertain differentiated tissue types such as meristems, Cartha et al.,Can. J. Bot. 59:1671-1679 (1981), hypocotyl sections, Cameya et al.,Plant Science Letters 21: 289-294 (1981), and stem node segments, Sakaet al., Plant Science Letters, 19: 193-201 (1980); Cheng et al., PlantScience Letters, 19: 91-99 (1980). Regeneration of whole sexually maturesoybean plants from somatic embryos generated from explants of immaturesoybean embryos has been reported (Ranch et al., In Vitro Cellular &Developmental Biology 21: 653-658 (1985). Regeneration of mature soybeanplants from tissue culture by organogenesis and embryogenesis has alsobeen reported (Barwale et al., Planta 167: 473-481 (1986); Wright etal., Plant Cell Reports 5: 150-154 (1986)).

Agents

One skilled in the art can refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Current Protocols in Molecular BiologyAusubel, et al., eds., John Wiley & Sons, N.Y. (1989), and supplementsthrough September (1998), Molecular Cloning, A Laboratory Manual,Sambrook et al., 2^(nd) Ed., Cold Spring Harbor Press, Cold SpringHarbor, N.Y. (1989), Genome Analysis: A Laboratory Manual 1: AnalyzingDNA, Birren et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1997); Genome Analysis: A Laboratory Manual 2: Detecting Genes, Birrenet al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1998);Genome Analysis: A Laboratory Manual 3: Cloning Systems, Birren et al.,Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1999); GenomeAnalysis: A Laboratory Manual 4: Mapping Genomes, Birren et al., ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1999); Plant MolecularBiology: A Laboratory Manual, Clark, Springer-Verlag, Berlin, (1997),Methods in Plant Molecular Biology, Maliga et al., Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1995). These texts can, of course, alsobe referred to in making or using an aspect of the invention. It isunderstood that any of the agents of the invention can be substantiallypurified and/or be biologically active and/or recombinant.

(a) Nucleic Acid Molecules

Nucleic acid molecules of the present invention include, withoutlimitation, nucleic acid molecules having a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1-1096 and complementsthereof. A subset of the nucleic acid molecules of the present inventionincludes nucleic acid molecules that encode a protein or fragmentthereof. Another subset of the nucleic acid molecules of the presentinvention are cDNA molecules. Another subset of the nucleic acidmolecules of the present invention includes nucleic acid molecules thatare marker molecules. A further subset of the nucleic acid molecules ofthe present invention are those nucleic acid molecules having promotersequences.

Fragment nucleic acid molecules may comprise significant portion(s) of,or indeed most of, these nucleic acid molecules. In preferredembodiments, the fragments may comprise smaller polynucleotides, e.g.,oligonucleotides having from about 20 to about 250 nucleotide residuesand more preferably, about 20 to about 100 nucleotide residues, or about40 to about 60 nucleotide residues. In another preferred embodiment,fragment molecules may be at least 15 nucleotides, at least 30nucleotides, at least 50 nucleotides, or at least 100 nucleotides.

The term “substantially purified,” as used herein, refers to a moleculeseparated from substantially all other molecules normally associatedwith it in its native state. More preferably a substantially purifiedmolecule is the predominant species present in a preparation. Asubstantially purified molecule may be greater than 60% free, preferably75% free, more preferably 90% free, and most preferably 95% free fromthe other molecules (exclusive of solvent) present in the naturalmixture. The term “substantially purified” is not intended to encompassmolecules present in their native state.

The agents of the present invention will preferably be “biologicallyactive” with respect to either a structural attribute, such as thecapacity of a nucleic acid to hybridize to another nucleic acidmolecule, or the ability of a protein to be bound by an antibody (or tocompete with another molecule for such binding). Alternatively, such anattribute may be catalytic and thus involve the capacity of the agent tomediate a chemical reaction or response.

The agents of the present invention may also be recombinant. As usedherein, the term recombinant describes (a) nucleic acid molecules thatare constructed or modified outside of cells and that can replicate orfunction in a living cell, (b) molecules that result from thetranscription, replication or translation of recombinant nucleic acidmolecules, or (c) organisms that contain recombinant nucleic acidmolecules or are modified using recombinant nucleic acid molecules.

It is understood that the agents of the present invention may be labeledwith reagents that facilitate detection of the agent, e.g., fluorescentlabels, (Prober et al., Science 238:336-340 (1987); Albarella et al., EP144914), chemical labels, (Sheldon et al., U.S. Pat. No. 4,582,789;Albarella et al., U.S. Pat. No. 4,563,417), and modified bases, (Miyoshiet al., EP 119448) including nucleotides with radioactive elements,e.g., ³²P, ³³P, ³⁵S or ¹²⁵I, such as ³²P dCTP.

It is further understood, that the present invention providesrecombinant bacterial, animal, fungal and plant cells and viralconstructs comprising the agents of the present invention.

Nucleic acid molecules or fragments thereof of the present invention arecapable of specifically hybridizing to other nucleic acid moleculesunder certain circumstances. As used herein, two nucleic acid moleculesare said to be capable of specifically hybridizing to one another if thetwo molecules are capable of forming an anti-parallel, double-strandednucleic acid structure. A nucleic acid molecule is said to be the“complement” of another nucleic acid molecule if they exhibit “completecomplementarity,” i.e., each nucleotide in one sequence is complementaryto its base pairing partner nucleotide in another sequence. Twomolecules are said to be “minimally complementary” if they can hybridizeto one another with sufficient stability to permit them to remainannealed to one another under at least conventional “low-stringency”conditions. Similarly, the molecules are said to be “complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under conventional“high-stringency” conditions. Nucleic acid molecules which hybridize toother nucleic acid molecules, e.g., at least under low stringencyconditions are said to be “hybridizable cognates” of the other nucleicacid molecules. Conventional stringency conditions are described bySambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed., ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1989) and by Haymes etal., Nucleic Acid Hybridization, A Practical Approach, IRL Press,Washington, D.C. (1985). Departures from complete complementarity aretherefore permissible, as long as such departures do not completelypreclude the capacity of the molecules to form a double-strandedstructure. Thus, in order for a nucleic acid molecule to serve as aprimer or probe it need only be sufficiently complementary in sequenceto be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed.

Appropriate stringency conditions which promote DNA hybridization, forexample, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or either the temperature or the salt concentration may be heldconstant while the other variable is changed.

In a preferred embodiment, a nucleic acid of the present invention willspecifically hybridize to one or more of the nucleic acid molecules setforth in SEQ ID NO: 1 through SEQ ID NO: 1096 or complements thereofunder moderately stringent conditions, for example at about 2.0×SSC andabout 65° C.

In a particularly preferred embodiment, a nucleic acid of the presentinvention will include those nucleic acid molecules that specificallyhybridize to one or more of the nucleic acid molecules set forth in SEQID NO: 1 through SEQ ID NO: 1096 or complements thereof under highstringency conditions such as 0.2×SSC and about 65° C.

In one aspect of the present invention, the nucleic acid molecules ofthe present invention comprise one or more of the nucleic acid sequencesset forth in SEQ ID NO: 1 through SEQ ID NO: 1096 or complements thereofor fragments of either. In another aspect of the present invention, oneor more of the nucleic acid molecules of the present invention share atleast 60% sequence identity with one or more of the nucleic acidsequences set forth in SEQ ID NO: 1 through SEQ ID NO: 1096 orcomplements thereof or fragments of either. In a further aspect of thepresent invention, one or more of the nucleic acid molecules of thepresent invention share at least 70% or more, e.g., at least 80%,sequence identity with one or more of the nucleic acid sequences setforth in SEQ ID NO: 1 through SEQ ID NO: 1096 or complements thereof orfragments of either. In a more preferred aspect of the presentinvention, one or more of the nucleic acid molecules of the presentinvention share at least 90% or more, e.g., at least 95% and up to 100%sequence identity with one or more of the nucleic acid sequences setforth in SEQ ID NO: 1 through SEQ ID NO: 1096 complements thereof orfragments of either.

As used herein “sequence identity” refers to the extent to which twooptimally aligned polynucleotide or peptide sequences are invariantthroughout a window of alignment of components, e.g., nucleotides oramino acids. An “identity fraction” for aligned segments of a testsequence and a reference sequence is the number of identical componentswhich are shared by the two aligned sequences divided by the totalnumber of components in reference sequence segment, i.e., the entirereference sequence or a smaller defined part of the reference sequence.“Percent identity” is the identity fraction times 100.

Useful methods for determining sequence identity are disclosed in Guideto Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego,1994, and Carillo, H., and Lipton, D., SIAM J Applied Math (1988)48:1073. More particularly, preferred computer programs for determiningsequence identity include the Basic Local Alignment Search Tool (BLAST)programs which are publicly available from National Center BiotechnologyInformation (NCBI) at the National Library of Medicine, NationalInstitute of Health, Bethesda, Md. 20894; see BLAST Manual, Altschul etal., NCBI, NLM, NIH; Altschul et al., J. Mol. Biol. 215:403-410 (1990);version 2.0 or higher of BLAST programs allows the introduction of gaps(deletions and insertions) into alignments; BLASTX can be used todetermine sequence identity between a polynucleotide sequence query anda protein sequence database; and, BLASTN can be used to determinesequence identity between between sequences.

For purposes of this invention “percent identity” shall be determinedusing BLASTX version 2.0.14 (default parameters), BLASTN version 2.0.14,or BLASTP 2.0.14.

A particularly preferred group of nucleic acid sequences are thosepresent in the soybean insert of the clones set forth in table 6 below.TABLE 6 Names of Clones Containing the Specified Gene Line Rhg4rhg1/frag 1 rhg1/frag 2 Forrest Forrest 1 Forrest 7 Forrest13 PekingPeking 1 Peking 7 Peking 13 Pickett Pickett 1 Pickett 7 Pickett 13PI84751 PI 84751.1 PI 84751.7 PI 84751.13 PI87631 PI 87631.1 PI 87631.7PI 87631.13 PI87631-1 PI 87631-1.1 PI 87631-1.13 PI88788R PI88788R.1 PI88788R.7 PI 88788R.13 PI89772 PI 89772.13 PI90763 PI 90763.7 PI 90763.13PI200499 PI 200499.1 PI 200499.7 PI 200499.13 PI209332 PI 209332.1 PI209332.13 PI404166 PI 404166.1 PI 404166.7 PI 404166.13 PI404198A PI404198A.7 PI 404198A.13 PI404198B PI 404198B.1 PI 404198B.7 PI404198B.13PI437654 PI 437654.1 PI 437654.7 PI 437654.13 PI438489B PI 438489.1 PI438489.7 PI 438489B.13 PI467312 PI 467312.1 PI 467312.7 PI 467312.13PI507354 PI 507354.1 PI 507354.7 PI 507354.13 PI423871 PI 423871.1 PI423871.7 PI 423871.13 PI407922 PI 407922.7 PI 407922.13 PI360843 PI360843.1 PI 360843.7 PI 360843.13 A2869 A2869.1 A2869.7 A2869.13 A2069A2069.1 A2069.13 Jack JACK1 JACK13 Will WILL1 WILL.7 WILL13 MinsoyMinsoy1 Minsoy.7 MINSOY13 Noir Noir1 Noir.7 NOIR13 Hutcheson Hutcheson1Hutcheson.7 Hutcheson.13 A1923 A1923.1 A1923.7 A1923.13 A2704 A2704.7A2704.13 Essex Essex1 Essex.7 ESSEX13 A3244 A3244.1 A3244.7 A3244.13Lee74 Lee74.1 Lee74.7 Lee74.13 PI437654 R107C17.7 R107C17.13

Table 5 shows clones comprising rhg1 and Rhg4 sequences. The “Lines”column indicates the cultivar from which the sequence in the clone isderived. The Rhg4, rhg1/frag1, and rhg1/frag2 columns show the clonesderived from the lines that have the Rhg4, rhg1 fragment 1, or rhg1fragment 2, respectively. Rhg4 is amplified with SEQ ID NOs: 48 and 49,which produces a 3.5 kb product. rhg1 fragment 1 is amplified with SEQID NOs: 24 and 25, which produces a 2.9 kb product, and rhg1 fragment 2is amplified with SEQ ID NOs: 26 and 27, which produces a 1.75 kbproduct. All fragments are subcloned into a pCR4-TOPO vector.

(i) Nucleic Acid Molecules Encoding Proteins or Fragments Thereof

A) rhg1

The present invention includes nucleic acid molecules that code for anrhg1 protein or fragment thereof. Examples of such nucleic acidmolecules include those that code for the proteins set forth in SEQ IDNOs: 1097, 1100, 1098, 1101, and 1102-1115. Examples of illustrativefragment molecules include, without limitation, an extracellular LRRdomain (rhg1, v.1, SEQ ID NO: 1097, residues 164-457; rhg1, v.2, SEQ IDNO: 1098, residues 141-434), a transmembrane domain (rhg1, v.1, SEQ IDNO: 1097, residues 508-530; rhg1, v.2, SEQ ID NO: 1098, residues 33-51and 485-507), and an STK domain (rhg1, v.1, SEQ ID NO: 1097, residues578-869; rhg1, v.2, SEQ ID NO: 1098, residues 555-846). Examples ofillustrative nucleic acid molecules include SEQ ID NOs: 5, 6, 8-23, and28-43.

B) Rhg4

The present invention includes nucleic acid molecules that code for anRhg4 protein or fragment thereof. Examples of such nucleic acidmolecules include those that code for the proteins set forth in SEQ IDNOs: 1099 and 1116-1119. Examples of illustrative fragment moleculesinclude, without limitation, an extracellular LRR domain (SEQ ID NO:1099, residues 34-44), a transmembrane domain (SEQ ID NO: 1099, residues449-471), and an STK domain (SEQ ID NO: 1099, residues 531-830).Examples of illustrative nucleic acid molecules include SEQ ID NOs: 7,44-47, and 50-53.

C) Rhg1 and Rhg4

In another further aspect of the present invention, nucleic acidmolecules of the present invention can comprise sequences which differfrom those encoding a protein or fragment thereof in SEQ ID NO: 1097through SEQ ID NO: 1119 due to fact that the different nucleic acidsequence encodes a protein having one or more conservative amino acidchanges. It is understood that codons capable of coding for suchconservative amino acid substitutions are known in the art.

It is well known in the art that one or more amino acids in a nativesequence can be substituted with another amino acid(s), the charge andpolarity of which are similar to that of the native amino acid, i.e., aconservative amino acid substitution. Conserved substitutions for anamino acid within the native polypeptide sequence can be selected fromother members of the class to which the naturally occurring amino acidbelongs. Amino acids can be divided into the following four groups: (1)acidic amino acids, (2) basic amino acids, (3) neutral polar aminoacids, and (4) neutral nonpolar amino acids. Representative amino acidswithin these various groups include, but are not limited to: (1) acidic(negatively charged) amino acids such as aspartic acid and glutamicacid; (2) basic (positively charged) amino acids such as arginine,histidine, and lysine; (3) neutral polar amino acids such as glycine,serine, threonine, cysteine, cystine, tyrosine, asparagine, andglutamine; and (4) neutral nonpolar (hydrophobic) amino acids such asalanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine.

Conservative amino acid changes within the native polypeptides sequencecan be made by substituting one amino acid within one of these groupswith another amino acid within the same group. Biologically functionalequivalents of the proteins or fragments thereof of the presentinvention can have ten or fewer conservative amino acid changes, morepreferably seven or fewer conservative amino acid changes, and mostpreferably five or fewer conservative amino acid changes. The encodingnucleotide sequence will thus have corresponding base substitutions,permitting it to encode biologically functional equivalent forms of theproteins or fragments of the present invention.

It is understood that certain amino acids may be substituted for otheramino acids in a protein structure without appreciable loss ofinteractive binding capacity with structures such as, for example,antigen-binding regions of antibodies or binding sites on substratemolecules. Because it is the interactive capacity and nature of aprotein that defines that protein's biological functional activity,certain amino acid sequence substitutions can be made in a proteinsequence and, of course, its underlying DNA coding sequence and,nevertheless, obtain a protein with like properties. It is thuscontemplated by the inventors that various changes may be made in thepeptide sequences of the proteins or fragments of the present invention,or corresponding DNA sequences that encode said peptides, withoutappreciable loss of their biological utility or activity. It isunderstood that codons capable of coding for such amino acid changes areknown in the art.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biological function on a protein is generallyunderstood in the art (Kyte and Doolittle, J. Mol. Biol. 157, 105-132(1982). It is accepted that the relative hydropathic character of theamino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like. In making such changes, the substitution ofamino acids whose hydropathic indices are within ±2 is preferred, thosewhich are within ±1 are particularly preferred, and those within ±0.5are even more particularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as govern by the hydrophilicity of its adjacent amino acids,correlates with a biological property of the protein. In a furtheraspect of the present invention, one or more of the nucleic acidmolecules of the present invention differ in nucleic acid sequence fromthose encoding a peptide set forth in SEQ ID NO: 1097 through SEQ ID NO:1119 or fragment thereof due to the fact that one or more codonsencoding an amino acid has been substituted for a codon that encodes anonessential substitution of the amino acid originally encoded.

Agents of the invention include nucleic acid molecules that encode atleast about a contiguous 10 amino acid region of a protein of thepresent invention, more preferably at least about a contiguous 11 to 14or larger amino acid region of a protein of the present invention. It isunderstood that the present invention includes nucleic acid moleculesthat specifically hybridize or exhibit a particular identity to thenucleic acid molecules described in (i). See (a) above.

(ii) Nucleic Acid Molecule Markers and Collections of Such Molecules

One aspect of the present invention concerns nucleic acid molecules ofthe present invention that can act as markers. As used herein, a“marker” is an indicator for the presence of at least one phenotype orpolymorphism, such as single nucleotide polymorphisms (SNPs), cleaveableamplified polymorphic sequences (CAPS), amplified fragment lengthpolymorphisms (AFLPs), restriction fragment length polymorphisms(RFLPs), simple sequence repeats (SSRs), or random amplified polymorphicDNA (RAPDs). A “nucleic acid marker” as used herein means a nucleic acidmolecule that is capable of being a marker for detecting a polymorphismor phenotype.

In one embodiment of the present invention, the nucleic acid markerspecifically hybridizes to a nucleic acid molecule having a nucleic acidsequence selected from the group SEQ NOs: 1-1096 and complementsthereof. In a preferred embodiment, the nucleic acid marker is capableof detecting an rhg1 SNP or INDEL set forth in table 2. In a preferredembodiment, the nucleic acid marker is capable of detecting an Rgh4 SNPor INDEL set forth in table 4. In another preferred embodiment thenucleic acid marker is a nucleic acid molecule capable of acting as aPCR primer to amplify an rhg1 or Rhg4 coding region. Examples of suchprimers include, without limitation, nucleic acid molecules having anucleic acid sequence set forth in SEQ ID NO: 401-1096 and complementsthereof. Such primers can be used in pairs to amplify a region(amplicons, e.g., without limitation, SEQ ID NOs: 54-400) that can befurther investigated using techniques known in the art such as nucleicacid sequencing. Preferred pairs are those with identical “Seq ID” (seeDescription of the Sequence Listing) except for the fact that one “SeqID” recites forward primer and one recites reverse primer.

In another embodiment of the present invention, the nucleic acid markerspecifically hybridizes to a nucleic acid molecule having a sequencethat is present on linkage group G within 500 kb or 100 kb, morepreferably within 50 kb, even more preferably within 25 kb of an rhg1allele, where the Rgh4 allele is preferably a sensitive allele, and morepreferably a sensitive allele from A3244. In a preferred embodiment thenucleic acid marker specifically hybridizes to M5 a nucleic acidmolecule having a sequence that is present on linkage group A2 within500 kb or 100 kb, more preferably within 50 kb, even more preferablywithin 25 kb of an Rhg4 allele, where the Rgh4 allele is preferably asensitive allele, and more preferably a sensitive allele from A3244.

As used herein, a “collection of nucleic acid molecules” is a populationof nucleic acid molecules where at least two, preferably all, of thenucleic acid molecules differ, at least in part, in their nucleic acidsequence. It is understood, that as used herein, an individual specieswithin a collection of nucleic acid molecules may be physically separateor alternatively not physically separate from one or more other specieswithin the collection of nucleic acid molecules. An example of asituation where individual species may be physically separate butconsidered a collection of nucleic acid molecules is where more than twospecies are present in a single location such as an array.

As used herein, where a collection of nucleic acid molecules is a markerfor a particular attribute, the level, pattern, occurrence and/orabsence of the nucleic acid molecules associated with the attribute arenot required to be the same between species of the collection. Forexample, the increase in the level of a species when in combination withthe decrease in a second species could be diagnostic for a particularattribute. In a preferred embodiment of the present invention, thelevel, pattern, occurrence and/or absence of a nucleic acid moleculeand/or collection of nucleic acid molecules of the present invention isa marker for SCN resistance.

In one embodiment, the marker is any nucleic acid molecule thatspecifically hybridizes to any nucleic acid sequence set forth herein.In another embodiment, the marker is a marker capable of distinguishingamong the haplotypes of either rhg1 or Rhg4. In yet another embodiment,more than one marker is used to simultaneously distinguish more than onehaplotype. In a preferred embodiment, two, three, four, six, eight,twenty five or fifty or more nucleic acid markers are usedsimultaneously. In another embodiment, one or more markers that arecapable of distinguishing among the haplotypes of rhg1 and one or moremarkers that are capable of distinguishing among the haplotypes of Rhg4are used together.

(iii) Nucleic Acid Molecules Having Promoter Sequences and OtherRegulatory Sequences

The present invention includes nucleic acid molecules that are an rhg1or Rhg4 promoter or fragment thereof. Examples of such nucleic acidmolecules include those set forth in SEQ ID NO: 2, upstream ofcoordinate 45163 and SEQ ID NO: 3, upstream of coordinate 46798. As usedherein a promoter is a nucleic acid sequence that when joined with acoding region is capable of expressing the protein or fragment thereofso encoded. In a preferred embodiment the promoter sequence correspondsto between 500 nucleotides and 5,000 nucleotides or between 300nucleotides and 700 nucleotides of the nucleic acid sequence set forthin SEQ ID NO: 2 between coordinates 45163 and 40163, or SEQ ID NO:3between coordinates 46798 and 41798, or the nucleic acid sequence setforth in SEQ ID NO: 4 between coordinates 111805 and 106805 Preferredpartial promoter regions include the TATA box region, e.g. atcoordinates 44234 through 44246 of SEQ ID NO: 2 and at coordinates107826 through 107835 of SEQ ID NO: 4, and CAAT box region, e.g. atcoordinates 106243 through 106259 of SEQ ID NO: 4.

Other regulatory sequences include introns or 3′ untranslated regions(3'UTRs) associated with rhg1 and Rhg4. In a preferred embodiment, anintron is selected from a nucleic acid comprising a sequence selectedfrom SEQ ID NO: 2 (rhg1 v.1 at coordinates 45315-45449, 45510-46940, and48764-48974), SEQ ID NO: 3 (rhg1 v.2 at coordinates 48764-48974) and SEQID NO: 4 (Rhg4 at coordinates 113969-114683). In another preferredembodiment, a 3'UTR is located within 5,000 nucleotides, more preferablewithin 1000 nucleotides in the 3′ direction of the last codingnucleotide of either rhg1 or Rhg4 (SEQ ID NO: 2, rhg1 v.1, coordinate49573, SEQ ID NO: 3, rhg1, v.2, coordinate 49573, SEQ ID NO: 4, Rhg4,coordinate 115204).

It is understood that the present invention includes nucleic acidmolecules that specifically hybridize or exhibit a particular identityto the nucleic acid molecules described in (iii). See (a) above.

(b) Protein and Peptide Molecules

A class of agents comprises one or more of the protein or peptidemolecules encoded by SEQ ID NO: 1097 through SEQ ID NO: 1119 or one ormore of the protein or fragment thereof or peptide molecules encoded byother nucleic acid agents of the present invention. As used herein, theterm “protein molecule” and “peptide molecule” mean any protein orprotein fragment or peptide or polypeptide molecule that comprises tenor more amino acids, preferably at least 11 or 12 or more, morepreferably at least 13 or 14 amino acids. It is well know in the artthat proteins may undergo modification, including post-translationalmodifications, such as, but not limited to, disulfide bond formation,glycosylation, phosphorylation, or oligomerization. Thus, as usedherein, the terms “protein molecule” and “peptide molecule” includemolecules that are modified by any biological or non-biological process.The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-amino acids. This definition is meant to include norleucine,ornithine, homocysteine, and homoserine.

One or more of the protein or peptide molecules may be produced viachemical synthesis, or more preferably, by expression in a suitablebacterial or eukaryotic host. Suitable methods for expression aredescribed by Sambrook, et al., (In: Molecular Cloning, A LaboratoryManual, 2nd Edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1989), or similar texts.

Another class of agents comprise protein or peptide molecules encoded bySEQ ID NO: 1097 through SEQ ID NO: 1119 or complements thereof or,fragments or fusions thereof in which non-essential, or not relevant,amino acid residues have been added, replaced, or deleted. An example ofsuch a homolog is a protein homolog of each soybean species, includingbut not limited to alfalfa, barley, Brassica, broccoli, cabbage, citrus,garlic, oat, oilseed rape, onion, canola, flax, pea, peanut, pepper,potato, rye, soybean, strawberry, sugarcane, sugarbeet, soybean, maize,rice, cotton, sorghum, Arabidopsis, wheat, pine, fir, eucalyptus, apple,lettuce, peas, lentils, grape, banana, tea, turf grasses, etc.Particularly preferred non-soybean plants to utilize for the isolationof homologs would include alfalfa, barley, oat, oilseed rape, canola,ornamentals, sugarcane, sugarbeet, soybean, maize, rice, cotton,sorghum, Arabidopsis, wheat, potato, and turf grasses. Such a homologcan be obtained by any of a variety of methods. Most preferably, asindicated above, one or more of the disclosed sequences (SEQ ID NO: 1through SEQ ID NO: 1096 or complements thereof) will be used to define apair of primers that may be used to isolate the protein homolog-encodingnucleic acid molecules from any desired species. Such molecules can beexpressed to yield protein homologs by recombinant means.

(c) Plant Constructs and Plant Transformants

One or more of the nucleic acid molecules of the invention may be usedin plant transformation or transfection. Exogenous genetic material maybe transferred into a plant cell and the plant cell regenerated into awhole, fertile or sterile plant. Exogenous genetic material is anygenetic material, whether naturally occurring or otherwise, from anysource that is capable of being inserted into any organism. In apreferred embodiment the exogenous genetic material includes a nucleicacid molecule of the present invention, preferably a nucleic acidmolecule having at least 20 nucleotides of a sequence selected from thegroup consisting of SEQ ID NO: 1 through SEQ ID NO: 1096 and complementsthereof. In a preferred embodiment, the nucleic acid molecule codes fora protein or fragment thereof described in Section (i). In anotherpreferred embodiment, the nucleic acid molecule is a promoter orfragment thereof described in Section (iii).

Such genetic material may be transferred into either monocotyledons anddicotyledons including, but not limited to tomato, eggplant, maize,soybean, Arabidopsis, phaseolus, peanut, alfalfa, wheat, rice, oat,sorghum, rye, tritordeum, millet, fescue, perennial ryegrass, sugarcane,cranberry, papaya, banana, banana, muskmelon, apple, cucumber,dendrobium, gladiolus, chrysanthemum, liliacea, cotton, eucalyptus,sunflower, canola, turfgrass, sugarbeet, coffee and dioscorea (Christou,In: Particle Bombardment for Genetic Engineering of Plants,Biotechnology Intelligence Unit. Academic Press, San Diego, Calif.(1996).

In a preferred embodiment, the genetic material is transferred to asoybean. Preferred soybeans to transfer an rhg1 SCN resistance alleleare selected from the group consisting of PI548402 (Peking), PI200499,A2869, Jack, A2069, PI209332 (No:4), PI404166 (Krasnoaarmejkaja),PI404198 (Sun huan do), PI437654 (Er-hej-jan), PI438489 (Chiquita),PI507354 (rokei 421), PI548655 (Forrest), PI548988 (Pickett), PI84751,PI437654, PI40792, Pyramid, Nathan, AG2201, A3469, AG3901, A3904,AG4301, AG4401, AG4501, AG4601, PION9492, PI88788, Dyer, Custer,Manokin, and Doles.

Preferred soybeans to transfer an Rhg4 SCN resistance allele areselected from the group consisting of PI548402 (Peking), PI437654(Er-hej-jan), PI438489 (Chiquita), PI507354 (Tokei 421), PI548655(Forrest), PI548988 (Pickett), PI88788, PI404198 (Sun Huan Do), PI404166(Krasnoaarmejkaja), Hartwig, Manokin, Doles, Dyer, and Custer.

Transfer of a nucleic acid that encodes for a protein can result inoverexpression of that protein in a transformed cell or transgenicplant. One or more of the proteins or fragments thereof encoded bynucleic acid molecules of the invention may be overexpressed in atransformed cell or transformed plant. Such overexpression may be theresult of transient or stable transfer of the exogenous geneticmaterial. Such overexpression can also result in SCN resistance to oneor more races of SCN.

Exogenous genetic material may be transferred into a host cell by theuse of a DNA vector or construct designed for such a purpose. Design ofsuch a vector is generally within the skill of the art (See, PlantMolecular Biology: A Laboratory Manual, Clark (ed.), Springier, New York(1997).

A construct or vector may include a plant promoter to express theprotein or protein fragment of choice. A number of promoters, which areactive in plant cells, have been described in the literature. Theseinclude the nopaline synthase (NOS) promoter (Ebert et al., Proc. Natl.Acad. Sci. (U.S.A.) 84:5745-5749 (1987), the octopine synthase (OCS)promoter (which are carried on tumor-inducing plasmids of Agrobacteriumtumefaciens), the caulimovirus promoters such as the cauliflower mosaicvirus (CaMV) 19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324(1987), and the CaMV 35S promoter (Odell et al., Nature 313:810-812(1985), the figwort mosaic virus 35S-promoter, the light-induciblepromoter from the small subunit of ribulose-1,5-bis-phosphatecarboxylase (ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl.Acad. Sci. (U.S.A.) 84:6624-6628 (1987), the sucrose synthase promoter(Yang et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:4144-4148 (1990), the Rgene complex promoter (Chandler et al., The Plant Cell 1:1175-1183(1989), and the chlorophyll a/b binding protein gene promoter, etc.These promoters have been used to create DNA constructs that have beenexpressed in plants; see, e.g., PCT publication WO 84/02913. The CaMV35S promoters are preferred for use in plants. Promoters known or foundto cause transcription of DNA in plant cells can be used in theinvention.

For the purpose of expression in source tissues of the plant, such asthe leaf, seed, root or stem, it is preferred that the promotersutilized have relatively high expression in these specific tissues.Tissue-specific expression of a protein of the present invention is aparticularly preferred embodiment. For this purpose, one may choose froma number of promoters for genes with tissue- or cell-specific or-enhanced expression. Examples of such promoters reported in theliterature include the chloroplast glutamine synthetase GS2 promoterfrom pea (Edwards et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:3459-3463(1990), the chloroplast fructose-1,6-biphosphatase (FBPase) promoterfrom wheat (Lloyd et al., Mol. Gen. Genet. 225:209-216 (1991), thenuclear photosynthetic ST-LS1 promoter from potato (Stockhaus et al.,EMBO J. 8:2445-2451 (1989), the STK (PAL) promoter and the glucoamylase(CHS) promoter from Arabidopsis thaliana. Also reported to be active inphotosynthetically active tissues are the ribulose-1,5-bisphosphatecarboxylase (RbcS) promoter from eastern larch (Larix laricina), thepromoter for the cab gene, cab6, from pine (Yamamoto et al., Plant CellPhysiol. 35:773-778 (1994), the promoter for the Cab-1 gene from wheat(Fejes et al., Plant Mol. Biol. 15:921-932 (1990), the promoter for theCAB-1 gene from spinach (Lubberstedt et al., Plant Physiol. 104:997-1006(1994), the promoter for the cab1R gene from rice (Luan et al., PlantCell. 4:971-981 (1992), the pyruvate, orthophosphate dikinase (PPDK)promoter from maize (Matsuoka et al., Proc. Natl. Acad. Sci. (U.S.A.)90: 9586-9590 (1993), the promoter for the tobacco Lhcb1*2 gene (Cerdanet al., Plant Mol. Biol. 33:245-255 (1997), the Arabidopsis thalianaSUC2 sucrose-H+ symporter promoter (Truemit et al., Planta. 196:564-570(1995), and the promoter for the thylakoid membrane proteins fromspinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS). Otherpromoters for the chlorophyll a/b-binding proteins may also be utilizedin the invention, such as the promoters for LhcB gene and PsbP gene fromwhite mustard (Sinapis alba; Kretsch et al., Plant Mol. Biol. 28:219-229(1995)).

For the purpose of expression in sink tissues of the plant, such as thetuber of the potato plant, the fruit of tomato, or the seed of maize,wheat, rice and barley, it is preferred that the promoters utilized inthe invention have relatively high expression in these specific tissues.A number of promoters for genes with tuber-specific or -enhancedexpression are known, including the class I patatin promoter (Bevan etal., EMBO J. 8:1899-1906 (1986); Jefferson et al., Plant Mol. Biol.14:995-1006 (1990)), the promoter for the potato tuber ADPGPP genes,both the large and small subunits, the sucrose synthase promoter(Salanoubat and Belliard, Gene 60:47-56 (1987), Salanoubat and Belliard,Gene 84:181-185 (1989)), the promoter for the major tuber proteinsincluding the 22 kd protein complexes and proteinase inhibitors(Hannapel, Plant Physiol. 101:703-704 (1993)), the promoter for thegranule bound starch synthase gene (GBSS) (Visser et al., Plant Mol.Biol. 17:691-699 (1991)), and other class I and II patatins promoters(Koster-Topfer et al., Mol Gen Genet. 219:390-396 (1989); Mignery etal., Gene. 62:27-44 (1988)).

Other promoters can also be used to express a protein or fragmentthereof in specific tissues, such as seeds or fruits. The promoter forβ-conglycinin (Chen et al., Dev. Genet. 10: 112-122 (1989)) or otherseed-specific promoters such as the napin and phaseolin promoters, canbe used. The zeins are a group of storage proteins found in maizeendosperm. Genomic clones for zein genes have been isolated (Pedersen etal., Cell 29:1015-1026 (1982)) and the promoters from these clones,including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD and genes, could also beused. Other promoters known to function, for example, in maize includethe promoters for the following genes: waxy, Brittle, Shrunken 2,Branching enzymes I and II, starch synthases, debranching enzymes,oleosins, glutelins and sucrose synthases. A particularly preferredpromoter for maize endosperm expression is the promoter for the glutelingene from rice, more particularly the Osgt-1 promoter (Zheng et al.,Mol. Cell Biol. 13:5829-5842 (1993)). Examples of promoters suitable forexpression in wheat include those promoters for the ADPglucosepyrosynthase (ADPGPP) subunits, the granule bound and other starchsynthase, the branching and debranching enzymes, theembryogenesis-abundant proteins, the gliadins and the glutenins.Examples of such promoters in rice include those promoters for theADPGPP subunits, the granule bound and other starch synthase, thebranching enzymes, the debranching enzymes, sucrose synthases and theglutelins. A particularly preferred promoter is the promoter for riceglutelin, Osgt-1. Examples of such promoters for barley include thosefor the ADPGPP subunits, the granule bound and other starch synthase,the branching enzymes, the debranching enzymes, sucrose synthases, thehordeins, the embryo globulins and the aleurone specific proteins.

Root specific promoters may also be used. An example of such a promoteris the promoter for the acid chitinase gene (Samac et al., Plant Mol.Biol. 25:587-596 (1994)). Expression in root tissue could also beaccomplished by utilizing the root specific subdomains of the CaMV35Spromoter that have been identified (Lam et al., Proc. Natl. Acad. Sci.(U.S.A.) 86:7890-7894 (1989)). Other root cell specific promotersinclude those reported by Conkling et al. (Conkling et al., PlantPhysiol. 93:1203-1211 (1990)).

Additional promoters that may be utilized are described, for example, inU.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147; 5,447,858; 5,608,144;5,608,144; 5,614,399; 5,633,441; 5,633,435; and 4,633,436. In addition,a tissue specific enhancer may be used (From et al., The Plant Cell1:977-984 (1989)).

Preferred promoters are those set forth in Section (a)(iii) of Agents.

Constructs or vectors may also include, with the coding region ofinterest, a nucleic acid sequence that acts, in whole or in part, toterminate transcription of that region. A number of such sequences havebeen isolated, including the Tr7 3′ sequence and the NOS 3′ sequence(Ingelbrecht et al., The Plant Cell 1:671-680 (1989); Bevan et al.,Nucleic Acids Res. 11:369-385 (1983)).

A vector or construct may also include regulatory elements. Examples ofsuch include the Adh intron 1 (Callis et al., Genes and Develop.1:1183-1200 (1987)), the sucrose synthase intron (Vasil et al., PlantPhysiol. 91:1575-1579 (1989)) and the TMV omega element (Gallie et al.,The Plant Cell 1:301-311 (1989)). These and other regulatory elementsmay be included when appropriate.

A vector or construct may also include a selectable marker. Selectablemarkers may also be used to select for plants or plant cells thatcontain the exogenous genetic material. Examples of such include, butare not limited to: a neomycin phosphotransferase gene (U.S. Pat. No.5,034,322), which codes for kanamycin resistance and can be selected forusing kanamycin, G418, etc.; a bar gene which codes for bialaphosresistance; genes which encode glyphosate resistance (U.S. Pat. Nos.4,940,835; 5,188,642; 4,971,908; 5,627,061); a nitrilase gene whichconfers resistance to bromoxynil (Stalker et al., J. Biol. Chem.263:6310-6314 (1988)); a mutant acetolactate synthase gene (ALS) whichconfers imidazolinone or sulphonylurea resistance (European PatentApplication 154,204 (Sep. 11, 1985)); and a methotrexate resistant DHFRgene (Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)).

A vector or construct may also include DNA sequence which encodes atransit peptide. Incorporation of a suitable chloroplast transit peptidemay also be employed (European Patent Application Publication Number0218571). Translational enhancers may also be incorporated as part ofthe vector DNA. DNA constructs could contain one or more 5′non-translated leader sequences which may serve to enhance expression ofthe gene products from the resulting mRNA transcripts. Such sequencesmay be derived from the promoter selected to express the gene or can bespecifically modified to increase translation of the mRNA. Such regionsmay also be obtained from viral RNAs, from suitable eukaryotic genes, orfrom a synthetic gene sequence. For a review of optimizing expression oftransgenes, see Koziel et al., Plant Mol. Biol. 32:393-405 (1996).

A vector or construct may also include a screenable marker. Screenablemarkers may be used to monitor expression. Exemplary screenable markersinclude: a β-glucuronidase or uidA gene (GUS) which encodes an enzymefor which various chromogenic substrates are known (Jefferson, PlantMol. Biol, Rep. 5:387-405 (1987); Jefferson et al., EMBO J. 6:3901-3907(1987)); an R-locus gene, which encodes a product that regulates theproduction of anthocyanin pigments (red color) in plant tissues(Dellaporta et al., Stadler Symposium 11:263-282 (1988)); a β-lactamasegene (Sutcliffe et al., Proc. Natl. Acad. Sci. (U.S.A.) 75:3737-3741(1978)), a gene which encodes an enzyme for which various chromogenicsubstrates are known (e.g., PADAC, a chromogenic cephalosporin); aluciferase gene (Ow et al., Science 234:856-859 (1986)); a xylE gene(Zukowsky et al., Proc. Natl. Acad. Sci. (U.S.A.) 80:1101-1105 (1983))which encodes a catechol dioxygenase that can convert chromogeniccatechols; an α-amylase gene (Ikatu et al., Bio/Technol. 8:241-242(1990)); a tyrosinase gene (Katz et al., J. Gen. Microbiol.129:2703-2714 (1983)) which encodes an enzyme capable of oxidizingtyrosine to DOPA and dopaquinone which in turn condenses to melanin; anα-galactosidase, which will turn a chromogenic α-galactose substrate.

Included within the terms “selectable or screenable marker genes” arealso genes which encode a secretable marker whose secretion can bedetected as a means of identifying or selecting for transformed cells.Examples include markers which encode a secretable antigen that can beidentified by antibody interaction, or even secretable enzymes which canbe detected catalytically. Secretable proteins fall into a number ofclasses, including small, diffusible proteins which are detectable,(e.g., by ELISA), small active enzymes which are detectable inextracellular solution (e.g., α-amylase, β-lactamase, phosphinothricintransferase), or proteins which are inserted or trapped in the cell wall(such as proteins which include a leader sequence such as that found inthe expression unit of extension or tobacco PR-S). Other possibleselectable and/or screenable marker genes will be apparent to those ofskill in the art.

There are many methods for introducing transforming nucleic acidmolecules into plant cells. Suitable methods are believed to includevirtually any method by which nucleic acid molecules may be introducedinto a cell, such as by Agrobacterium infection or direct delivery ofnucleic acid molecules such as, for example, by PEG-mediatedtransformation, by electroporation or by acceleration of DNA coatedparticles, etc (Potrykus, Ann. Rev. Plant Physiol. Plant Mol. Biol.42:205-225 (1991); Vasil, Plant Mol. Biol. 25:925-937 (1994)). Forexample, electroporation has been used to transform maize protoplasts(Fromm et al., Nature 312:791-793 (1986)).

Other vector systems suitable for introducing transforming DNA into ahost plant cell include but are not limited to binary artificialchromosome (BIBAC) vectors (Hamilton et al., Gene 200:107-116 (1997));and transfection with RNA viral vectors (Della-Cioppa et al., Ann. N.Y.Acad. Sci. (1996), 792 (Engineering Plants for Commercial Products andApplications), 57-61). Additional vector systems also include plantselectable YAC vectors such as those described in Mullen et al.,Molecular Breeding 4:449-457 (1988)).

Technology for introduction of DNA into cells is well known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb,Virology 54:536-539 (1973)); (2) physical methods such as microinjection(Capecchi, Cell 22:479-488 (1980)), electroporation (Wong and Neumann,Biochem. Biophys. Res. Commun. 107:584-587 (1982); Fromm et al., Proc.Natl. Acad. Sci. (U.S.A.) 82:5824-5828 (1985); U.S. Pat. No. 5,384,253);and the gene gun (Johnston and Tang, Methods Cell Biol. 43:353-365(1994)); (3) viral vectors (Clapp, Clin. Perinatol. 20:155-168 (1993);Lu et al., J. Exp. Med 178:2089-2096 (1993); Eglitis and Anderson,Biotechniques 6:608-614 (1988)); and (4) receptor-mediated mechanisms(Curiel et al., Hum. Gen. Ther. 3:147-154 (1992), Wagner et al., Proc.Natl. Acad. Sci. (USA) 89:6099-6103 (1992)).

Acceleration methods that may be used include, for example,microprojectile bombardment and the like. One example of a method fordelivering transforming nucleic acid molecules to plant cells ismicroprojectile bombardment. This method has been reviewed by Yang andChristou (eds.), Particle Bombardment Technology for Gene Transfer,Oxford Press, Oxford, England (1994)). Non-biological particles(microprojectiles) that may be coated with nucleic acids and deliveredinto cells by a propelling force. Exemplary particles include thosecomprised of tungsten, gold, platinum and the like.

A particular advantage of microprojectile bombardment, in addition to itbeing an effective means of reproducibly transforming monocots, is thatneither the isolation of protoplasts (Cristou et al., Plant Physiol.87:671-674 (1988)) nor the susceptibility of Agrobacterium infection arerequired. An illustrative embodiment of a method for delivering DNA intomaize cells by acceleration is a biolistics α-particle delivery system,which can be used to propel particles coated with DNA through a screen,such as a stainless steel or Nytex screen, onto a filter surface coveredwith corn cells cultured in suspension. Gordon-Kamm et al., describesthe basic procedure for coating tungsten particles with DNA (Gordon-Kammet al., Plant Cell 2:603-618 (1990)). The screen disperses the tungstennucleic acid particles so that they are not delivered to the recipientcells in large aggregates. A particle delivery system suitable for usewith the invention is the helium acceleration PDS-1000/He gun isavailable from Bio-Rad Laboratories (Bio-Rad, Hercules, Calif.)(Sanfordet al., Technique 3:3-16 (1991)).

For the bombardment, cells in suspension may be concentrated on filters.Filters containing the cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between the gun and thecells to be bombarded.

Alternatively, immature embryos or other target cells may be arranged onsolid culture medium. The cells to be bombarded are positioned at anappropriate distance below the microprojectile stopping plate. Ifdesired, one or more screens are also positioned between theacceleration device and the cells to be bombarded. Through the use oftechniques set forth herein one may obtain up to 1000 or more foci ofcells transiently expressing a screenable or selectable marker gene. Thenumber of cells in a focus which express the exogenous gene product 48hours post-bombardment often range from one to ten and average one tothree.

In bombardment transformation, one may optimize the pre-bombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature embryos.

In another alternative embodiment, plastids can be stably transformed.Methods disclosed for plastid transformation in higher plants includethe particle gun delivery of DNA containing a selectable marker andtargeting of the DNA to the plastid genome through homologousrecombination (Svab et al., Proc. Natl. Acad. Sci. (U.S.A.) 87:8526-8530(1990); Svab and Maliga, Proc. Natl. Acad. Sci. (U.S.A.) 90:913-917(1993); Staub and Maliga, EMBO J. 12:601-606 (1993); U.S. Pat. Nos.5,451,513 and 5,545,818).

Accordingly, it is contemplated that one may wish to adjust variousaspects of the bombardment parameters in small-scale studies to fullyoptimize the conditions. One may particularly wish to adjust physicalparameters such as gap distance, flight distance, tissue distance andhelium pressure. One may also minimize the trauma reduction factors bymodifying conditions which influence the physiological state of therecipient cells and which may therefore influence transformation andintegration efficiencies. For example, the osmotic state, tissuehydration and the subculture stage or cell cycle of the recipient cellsmay be adjusted for optimum transformation. The execution of otherroutine adjustments will be known to those of skill in the art in lightof the present disclosure.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedE15s plant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example the methods described by Fraley etal., Bio/Technology 3:629-635 (1985) and Rogers et al., Methods Enzymol.153:253-277 (1987). Further, the integration of the T-DNA is arelatively precise process resulting in few rearrangements. The regionof DNA to be transferred is defined by the border sequences andintervening DNA is usually inserted into the plant genome as described(Spielmann et al., Mol. Gen. Genet. 205:34 (1986)).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., In: Plant DNA InfectiousAgents, Hohn and Schell (eds.), Springer-Verlag, New York, pp. 179-203(1985)). Moreover, technological advances in vectors forAgrobacterium-mediated gene transfer have improved the arrangement ofgenes and restriction sites in the vectors to facilitate construction ofvectors capable of expressing various polypeptide coding genes. Thevectors described have convenient multi-linker regions flanked by apromoter and a polyadenylation site for direct expression of insertedpolypeptide coding genes and are suitable for present purposes (Rogerset al., Methods Enzymol. 153:253-277 (1987)). In addition, Agrobacteriumcontaining both armed and disarmed Ti genes can be used for thetransformations. In those plant strains where Agrobacterium-mediatedtransformation is efficient, it is the method of choice because of thefacile and defined nature of the gene transfer.

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene. Morepreferred is a transgenic plant that is homozygous for the addedstructural gene; i.e., a transgenic plant that contains two added genes,one gene at the same locus on each chromosome of a chromosome pair. Ahomozygous transgenic plant can be obtained by sexually mating (selfing)an independent segregant transgenic plant that contains a single addedgene, germinating some of the seed produced and analyzing the resultingplants produced for the gene of interest.

It is also to be understood that two different transgenic plants canalso be mated to produce offspring that contain two independentlysegregating, exogenous genes. Selfing of appropriate progeny can produceplants that are homozygous for both added, exogenous genes that encode apolypeptide of interest. Backcrossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated, as isvegetative propagation.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation and combinations of these treatments (See, for example,Potrykus et al., Mol. Gen. Genet. 205:193-200 (1986); Lorz et al., Mol.Gen. Genet. 199:178 (1985); Fromm et al., Nature 319:791 (1986);Uchimiya et al., Mol. Gen. Genet. 204:204 (1986); Marcotte et al.,Nature 335:454-457 (1988)).

Application of these systems to different plant strains depends upon theability to regenerate that particular plant strain from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (Fujimura et al., Plant Tissue Culture Letters 2:74(1985); Toriyama et al., Theor Appl. Genet. 205:34 (1986); Yamada etal., Plant Cell Rep. 4:85 (1986); Abdullah et al., Biotechnology 4:1087(1986)).

To transform plant strains that cannot be successfully regenerated fromprotoplasts, other ways to introduce DNA into intact cells or tissuescan be utilized. For example, regeneration of cereals from immatureembryos or explants can be effected as described (Vasil, Biotechnology6:397 (1988)). In addition, “particle gun” or high-velocitymicroprojectile technology can be utilized (Vasil et al., Bio/Technology10:667 (1992)).

Using the latter technology, DNA is carried through the cell wall andinto the cytoplasm on the surface of small metal particles as described(Klein et al., Nature 328:70 (1987); Klein et al., Proc. Natl. Acad.Sci. (U.S.A.) 85:8502-8505 (1988); McCabe et al., Bio/Technology 6:923(1988)). The metal particles penetrate through several layers of cellsand thus allow the transformation of cells within tissue explants.

The regeneration, development and cultivation of plants from singleplant protoplast transformants or from various transformed explants arewell known in the art (Weissbach and Weissbach, In: Methods for PlantMolecular Biology, Academic Press, San Diego, Calif., (1988)). Thisregeneration and growth process typically includes the steps ofselection of transformed cells, culturing those individualized cellsthrough the usual stages of embryonic development through the rootedplantlet stage. Transgenic embryos and seeds are similarly regenerated.The resulting transgenic rooted shoots are thereafter planted in anappropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a protein of interest is well known in theart. Preferably, the regenerated plants are self-pollinated to providehomozygous transgenic plants. Otherwise, pollen obtained from theregenerated plants is crossed to seed-grown plants of agronomicallyimportant lines. Conversely, pollen from plants of these important linesis used to pollinate regenerated plants. A transgenic plant of theinvention containing a desired polypeptide is cultivated using methodswell known to one skilled in the art.

There are a variety of methods for the regeneration of plants from planttissue. The particular method of regeneration will depend on thestarting plant tissue and the particular plant species to beregenerated.

Methods for transforming dicots, primarily by use of Agrobacteriumtumefaciens and obtaining transgenic plants have been published forcotton (U.S. Pat. No. 5,004,863; U.S. Pat. Nos. 5,159,135; 5,518,908);soybean (U.S. Pat. No. 5,569,834; U.S. Pat. No. 5,416,011; McCabe etal., Biotechnology 6-923 (1988); Christou et al., Plant Physiol.87:671-674 (1988)); Brassica (U.S. Pat. No. 5,463,174); peanut (Cheng etal., Plant Cell Rep. 15:653-657 (1996), McKently et al., Plant Cell Rep.14:699-703 (1995)); papaya; and pea (Grant et al., Plant Cell Rep.15:254-258 (1995)).

Transformation of monocotyledons using electroporation, particlebombardment and Agrobacterium have also been reported. Transformationand plant regeneration have been achieved in asparagus (Bytebier et al.,Proc. Natl. Acad. Sci. (USA) 84:5354 (1987)); barley (Wan and Lemaux,Plant Physiol 104:37 (1994)); maize (Rhodes et al., Science 240:204(1988); Gordon-Kamm et al., Plant Cell 2:603-618 (1990); Fromm et al.,Bio/Technology 8:833 (1990); Koziel et al., Bio/Technology 11:194(1993); Armstrong et al., Crop Science 35:550-557 (1995)); oat (Somerset al., Bio/Technology 10:1589 (1992)); orchard grass (Horn et al.,Plant Cell Rep. 7:469 (1988)); rice (Toriyama et al., Theor Appl. Genet.205:34 (1986); Part et al., Plant Mol. Biol. 32:1135-1148 (1996);Abedinia et al., Aust. J. Plant Physiol. 24:133-141 (1997); Zhang andWu, Theor. Appl. Genet. 76:835 (1988); Zhang et al., Plant Cell Rep.7:379 (1988); Battraw and Hall, Plant Sci. 86:191-202 (1992); Christouet al., Bio/Technology 9:957 (1991)); rye (De la Pena et al., Nature325:274 (1987)); sugarcane (Bower and Birch, Plant J. 2:409 (1992));tall fescue (Wang et al, Bio/Technology 10:691 (1992)) and wheat (Vasilet al., Bio/Technology 10:667 (1992); U.S. Pat. No. 5,631,152).

Assays for gene expression based on the transient expression of clonednucleic acid constructs have been developed by introducing the nucleicacid molecules into plant cells by polyethylene glycol treatment,electroporation, or particle bombardment (Marcotte et al., Nature335:454-457 (1988); Marcotte et al., Plant Cell 1:523-532 (1989);McCarty et al., Cell 66:895-905 (1991); Hattori et al., Genes Dev.6:609-618 (1992); Goff et al, EMBO J. 9:2517-2522 (1990)). Transientexpression systems may be used to functionally dissect gene constructs(see generally, Mailga et al., Methods in Plant Molecular Biology, ColdSpring Harbor Press (1995)).

Any of the nucleic acid molecules of the invention may be introducedinto a plant cell in a permanent or transient manner in combination withother genetic elements such as vectors, promoters, enhancers, etc.Further, any of the nucleic acid molecules of the invention may beintroduced into a plant cell in a manner that allows for overexpressionof the protein or fragment thereof encoded by the nucleic acid molecule.

Cosuppression is the reduction in expression levels, usually at thelevel of RNA, of a particular endogenous gene or gene family by theexpression of a homologous sense construct that is capable oftranscribing mRNA of the same strandedness as the transcript of theendogenous gene (Napoli et al., Plant Cell 2:279-289 (1990); van derKrol et al., Plant Cell 2:291-299 (1990)). Cosuppression may result fromstable transformation with a single copy nucleic acid molecule that ishomologous to a nucleic acid sequence found within the cell (Prolls andMeyer, Plant J. 2:465-475 (1992)) or with multiple copies of a nucleicacid molecule that is homologous to a nucleic acid sequence found withinthe cell (Mittlesten et al., Mol. Gen. Genet. 244:325-330 (1994)).Genes, even though different, linked to homologous promoters may resultin the cosuppression of the linked genes (Vaucheret, C.R. Acad. Sci. III316:1471-1483 (1993); Flavell, Proc. Natl. Acad. Sci. (U.S.A.)91:3490-3496 (1994)); van Blokland et al., Plant J. 6:861-877 (1994);Jorgensen, Trends Biotechnol. 8:340-344 (1990); Meins and Kunz, In: GeneInactivation and Homologous Recombination in Plants, Paszkowski (ed.),pp. 335-348, Kluwer Academic, Netherlands (1994)).

It is understood that one or more of the nucleic acids of the inventionmay be introduced into a plant cell and transcribed using an appropriatepromoter with such transcription resulting in the cosuppression of anendogenous protein.

Antisense approaches are a way of preventing or reducing gene functionby targeting the genetic material (U.S. Pat. Nos. 4,801,540 and5,107,065 Mol et al., FEBS Lett. 268:427-430 (1990)). The objective ofthe antisense approach is to use a sequence complementary to the targetgene to block its expression and create a mutant cell line or organismin which the level of a single chosen protein is selectively reduced orabolished. Antisense techniques have several advantages over other‘reverse genetic’ approaches. The site of inactivation and itsdevelopmental effect can be manipulated by the choice of promoter forantisense genes or by the timing of external application ormicroinjection. Antisense can manipulate its specificity by selectingeither unique regions of the target gene or regions where it shareshomology to other related genes (Hiatt et al., In: Genetic Engineering,Setlow (ed.), Vol. 11, New York: Plenum 49-63 (1989)).

The principle of regulation by antisense RNA is that RNA that iscomplementary to the target mRNA is introduced into cells, resulting inspecific RNA:RNA duplexes being formed by base pairing between theantisense substrate and the target mRNA (Green et al., Annu. Rev.Biochem. 55:569-597 (1986)). Under one embodiment, the process involvesthe introduction and expression of an antisense gene sequence. Such asequence is one in which part or all of the normal gene sequences areplaced under a promoter in inverted orientation so that the ‘wrong’ orcomplementary strand is transcribed into a noncoding antisense RNA thathybridizes with the target mRNA and interferes with its expression(Takayama and Inouye, Crit. Rev. Biochem. Mol. Biol. 25:155-184 (1990)).An antisense vector is constructed by standard procedures and introducedinto cells by transformation, transfection, electroporation,microinjection, infection, etc. The type of transformation and choice ofvector will determine whether expression is transient or stable. Thepromoter used for the antisense gene may influence the level, timing,tissue, specificity, or inducibility of the antisense inhibition.

It is understood that the activity of a protein in a plant cell may bereduced or depressed by growing a transformed plant cell containing anucleic acid molecule whose non-transcribed strand encodes a protein orfragment thereof.

Post transcriptional gene silencing (PTGS) can result in virus immunityor gene silencing in plants. PTGS is induced by dsRNA and is mediated byan RNA-dependent RNA polymerase, present in the cytoplasm, that requiresa dsRNA template. The dsRNA is formed by hybridization of complementarytransgene mRNAs or complementary regions of the same transcript. Duplexformation can be accomplished by using transcripts from one sense geneand one antisense gene co-located in the plant genome, a singletranscript that has self-complementarity, or sense and antisensetranscripts from genes brought together by crossing. The dsRNA-dependentRNA polymerase makes a complementary strand from the transgene mRNA andRNAse molecules attach to this complementary strand (cRNA). ThesecRNA-RNAse molecules hybridize to the endogene mRNA and cleave thesingle-stranded RNA adjacent to the hybrid. The cleaved single-strandedRNAs are further degraded by other host RNAses because one will lack acapped 5′ end and the other will lack a poly(A) tail (Waterhouse et al.,PNAS 95: 13959-13964 (1998)).

It is understood that one or more of the nucleic acids of the inventionmay be introduced into a plant cell and transcribed using an appropriatepromoter with such transcription resulting in the postranscriptionalgene silencing of an endogenous transcript.

Antibodies have been expressed in plants (Hiatt et al., Nature 342:76-78(1989); Conrad and Fielder, Plant Mol. Biol. 26:1023-1030 (1994)).Cytoplasmic expression of a scFv (single-chain Fv antibodies) has beenreported to delay infection by artichoke mottled crinkle virus.Transgenic plants that express antibodies directed against endogenousproteins may exhibit a physiological effect (Philips et al., EMBO J.16:4489-4496 (1997); Marion-Poll, Trends in Plant Science 2:447-448(1997)). For example, expressed anti-abscissic antibodies have beenreported to result in a general perturbation of seed development(Philips et al., EMBO J. 16: 4489-4496 (1997)).

Antibodies that are catalytic may also be expressed in plants (abzymes).The principle behind abzymes is that since antibodies may be raisedagainst many molecules, this recognition ability can be directed towardgenerating antibodies that bind transition states to force a chemicalreaction forward (Persidas, Nature Biotechnology 15:1313-1315 (1997);Baca et al., Ann. Rev. Biophys. Biomol. Struct. 26:461-493 (1997)). Thecatalytic abilities of abzymes may be enhanced by site directedmutagenesis. Examples of abzymes are, for example, set forth in U.S.Pat. No. 5,658,753; U.S. Pat. No. 5,632,990; U.S. Pat. No. 5,631,137;U.S. Pat. No. 5,602,015; U.S. Pat. No. 5,559,538; U.S. Pat. No.5,576,174; U.S. Pat. No. 5,500,358; U.S. Pat. No. 5,318,897; U.S. Pat.No. 5,298,409; U.S. Pat. No. 5,258,289 and U.S. Pat. No. 5,194,585.

It is understood that any of the antibodies of the invention may beexpressed in plants and that such expression can result in aphysiological effect. It is also understood that any of the expressedantibodies may be catalytic.

(d) Antibodies

One aspect of the present invention concerns antibodies, single-chainantigen binding molecules, or other proteins that specifically bind toone or more of the protein or peptide molecules of the present inventionand their homologues, fusions or fragments. Such antibodies may be usedto quantitatively or qualitatively detect the protein or peptidemolecules of the present invention. As used herein, an antibody orpeptide is said to “specifically bind” to a protein or peptide moleculeof the present invention if such binding is not competitively inhibitedby the presence of non-related molecules.

Nucleic acid molecules that encode all or part of the protein of thepresent invention can be expressed, via recombinant means, to yieldprotein or peptides that can in turn be used to elicit antibodies thatare capable of binding the expressed protein or peptide. Such antibodiesmay be used in immunoassays for that protein. Such protein-encodingmolecules, or their fragments may be a “fusion” molecule (i.e., a partof a larger nucleic acid molecule) such that, upon expression, a fusionprotein is produced. It is understood that any of the nucleic acidmolecules of the present invention may be expressed, via recombinantmeans, to yield proteins or peptides encoded by these nucleic acidmolecules.

The antibodies that specifically bind proteins and protein fragments ofthe present invention may be polyclonal or monoclonal and may compriseintact immunoglobulins, or antigen binding portions of immunoglobulinsfragments (such as (F(ab′), F(ab′)₂), or single-chain immunoglobulinsproducible, for example, via recombinant means. It is understood thatpractitioners are familiar with the standard resource materials whichdescribe specific conditions and procedures for the construction,manipulation and isolation of antibodies (see, for example, Harlow andLane, In: Antibodies: A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1988)).

Murine monoclonal antibodies are particularly preferred. BALB/c mice arepreferred for this purpose, however, equivalent strains may also beused. The animals are preferably immunized with approximately 25 kg ofpurified protein (or fragment thereof) that has been emulsified in asuitable adjuvant (such as TiterMax adjuvant (Vaxcel, Norcross, Ga.)).Immunization is preferably conducted at two intramuscular sites, oneintraperitoneal site and one subcutaneous site at the base of the tail.An additional i.v. injection of approximately 25 μg of antigen ispreferably given in normal saline three weeks later. After approximately11 days following the second injection, the mice may be bled and theblood screened for the presence of anti-protein or peptide antibodies.Preferably, a direct binding Enzyme-Linked Immunoassay (ELISA) isemployed for this purpose.

More preferably, the mouse having the highest antibody titer is given athird i.v. injection of approximately 25 μg of the same protein orfragment. The splenic leukocytes from this animal may be recovered 3days later and then permitted to fuse, most preferably, usingpolyethylene glycol, with cells of a suitable myeloma cell line (suchas, for example, the P3X63Ag8.653 myeloma cell line). Hybridoma cellsare selected by culturing the cells under “HAT”(hypoxanthine-aminopterin-thymine) selection for about one week. Theresulting clones may then be screened for their capacity to producemonoclonal antibodies (“mAbs”), preferably by direct ELISA.

In one embodiment, anti-protein or peptide monoclonal antibodies areisolated using a fusion of a protein or peptide of the presentinvention, or conjugate of a protein or peptide of the presentinvention, as immunogens. Thus, for example, a group of mice can beimmunized using a fusion protein emulsified in Freund's completeadjuvant (e.g., approximately 50 μg of antigen per immunization). Atthree week intervals, an identical amount of antigen is emulsified inFreund's incomplete adjuvant and used to immunize the animals. Ten daysfollowing the third immunization, serum samples are taken and evaluatedfor the presence of antibody. If antibody titers are too low, a fourthbooster can be employed. Polysera capable of binding the protein orpeptide can also be obtained using this method.

In a preferred procedure for obtaining monoclonal antibodies, thespleens of the above-described immunized mice are removed, disrupted andimmune splenocytes are isolated over a ficoll gradient. The isolatedsplenocytes are fused, using polyethylene glycol with BALB/c-derivedHGPRT (hypoxanthine guanine phosphoribosyl transferase) deficientP3×63xAg8.653 plasmacytoma cells. The fused cells are plated into 96well microtiter plates and screened for hybridoma fusion cells by theircapacity to grow in culture medium supplemented with hypothanthine,aminopterin and thymidine for approximately 2-3 weeks.

Hybridoma cells that arise from such incubation are preferably screenedfor their capacity to produce an immunoglobulin that binds to a proteinof interest. An indirect ELISA may be used for this purpose. In brief,the supernatants of hybridomas are incubated in microtiter wells thatcontain immobilized protein. After washing, the titer of boundimmunoglobulin can be determined using, for example, a goat anti-mouseantibody conjugated to horseradish peroxidase. After additional washing,the amount of immobilized enzyme is determined (for example through theuse of a chromogenic substrate). Such screening is performed as quicklyas possible after the identification of the hybridoma in order to ensurethat a desired clone is not overgrown by non-secreting neighbor cells.Desirably, the fusion plates are screened several times since the ratesof hybridoma growth vary. In a preferred sub-embodiment, a differentantigenic form may be used to screen the hybridoma. Thus, for example,the splenocytes may be immunized with one immunogen, but the resultinghybridomas can be screened using a different immunogen. It is understoodthat any of the protein or peptide molecules of the present inventionmay be used to raise antibodies.

Such antibody molecules or their fragments may be used for diagnosticpurposes. Where the antibodies are intended for diagnostic purposes, itmay be desirable to derivatize them, for example with a ligand group(such as biotin) or a detectable marker group (such as a fluorescentgroup, a radioisotope or an enzyme).

The ability to produce antibodies that bind the protein or peptidemolecules of the present invention permits the identification of mimeticcompounds of those molecules. A “mimetic compound” is a compound that isnot that compound, or a fragment of that compound, but which nonethelessexhibits an ability to specifically bind to antibodies directed againstthat compound.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1

In this example, DNA is extracted from soybean plants, amplified, andmapped.

A single trifoliate leaf is collected from the newest growth of fourweek old soybean plants. Leaf tissue from the leaf is placed on ice andstored at −80° C. The frozen tissue is lyophilized, and approximately0.01 grams of the tissue is used for DNA extraction. The 0.01 grams ofleaf tissue is ground to powder in 1.4 ml tubes. 600 microliters (1) ofDNA extraction buffer consisting of 0.5M NaCl, 0.1MTris-(hydroxymethyl)aminomethane pH 8.0, 0.05 Methylenediaminetetra-acetic acid (EDTA), 10.0 g L⁻¹ sodium dodecylsulfate (SDS), and 2 g L⁻¹ phenantroline (dissolved in 0.01 L ethanol)is heated to 65° C. (with 0.77 g L⁻¹ dithiothreitol added immediatelybefore use) is added to each tube, and each tube is mixed thoroughly.The samples are placed in a 65° C. water bath for 15 minutes and shakenby hand after 10 minutes. The samples are taken out of the water bathand cooled to room temperature, and then 200 μl of 5 M KOAc is added toeach tube. The samples are inverted and placed at 4° C. for 20 minutes.Samples are then centrifuged for 12 minutes at 6200×g and thesupernatant (about 600 μl) is transferred to new tubes. DNA isprecipitated with 330 μl of cold isopropanol and placed at −20° C. for 1hr. The DNA is pelleted by centrifuging at 6200×g for 10 minutes andwashed with 70% EtOH. The DNA is pelleted by centrifugation at 6200×gfor 10 minutes and dried using a Speed-Vac. The DNA is dissolved in 100μl of TE_(0.1) (0.01 M Tris-HCl pH 8.0, 0.0001 M EDTA). The extractionwill generally yield 500 ng DNA μl⁻¹.

A polymerase chain reaction (PCR) is conducted with 5 to 10 ng genomicDNA in 10 μl volumes of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.001%gelatin, 1.5 mM MgCl₂, 0.1 mM of each dNTP, 150 nM of each primer, 0.01mM Cresol Red, 2% sucrose and 0.32 units of AmpliTaq DNA Polymerase(Perkin Elmer Instruments Inc., USA). For thermocycling, the Gene AmpPCR System 9700 (Perkin Elmer Instruments Inc., USA) is used with onestep of 94° C. for 3 minutes, then 32 cycles of 94° C., 47° C., and 72°C. steps of 25 sec each and one final step of 72° C. for 3 minutes. ThePCR products are run on a 6% polyacrylamide gel (30 cm×8 cm×1 mm) in1×TAE (40 mM Tris-HCl, pH 8.3, 1 mM EDTA) at 180 v for 45 minutes. Thegels are stained using SYBR Gold (Molecular Probes, Eugene, Oreg.)according to the manufacturer's instructions.

SSR primer screening for polymorphism is performed using PIC, HS-1, Willand PI507354 genotypes. SSRs that are polymorphic and easy to score(i.e., clear banding pattern and good separation between alleles) aremapped using the HS-1×PIC (F2) and/or Will x PI507354 (RIL) mappingpopulations. At least one SSR per BAC sequence is mapped. DNA markersthat exhibited codominant banding patterns are scored as homozygous forone or the other parent or as heterozygous, exhibiting both parentalalleles. Marker scores are checked for segregation distortion using thechi-squared test for goodness of fit to expected ratios. Linkagerelationships are determined using Mapmaker Version 3.0b with a LOD of3.0 (Whitehead Institute, Cambridge, Mass.).

EXAMPLE 2

DNA fragments containing candidates for genes rhg1 and Rhg4 fromsusceptible and resistant soybean lines are subcloned into a TA cloningplasmid (TOPO TA Cloning Kit, Version E, Invitrogen Corporation, 1600Faraday Avenue, Carlsbad, Calif.).

Genomic DNA from 24 susceptible and 9 resistant lines is isolated usingstandard techniques. Approximately 500 nanograms (ng) of DNA is used forPCR amplification. Resistant BAC DNA is isolated by using AUTOGEN(AutoGen Corp., 35 Loring Drive Framingham, Mass.). PCR amplification isthen performed using 0.1-0.2 ng of resistant BAC DNA. The primers thatare used to amplify candidate rhg1 genes PCR are as follows:

Fragment I (2,892 bp) primer (SEQ ID NO: 25), GCA ATA CTT GAA GGA ATATGT CCA C; primer (SEQ ID NO: 24), beginning at start codon, ATG GAT GGTAAA AAT TCA AAA CTA AAC; modified reverse primer 1 (SEQ ID NO: 1123),beginning 5 bp before start codon; GTT GTA TGG ATG GTA AAA ATT CAA AAC.Fragment II (1,746 bp) reverse primer 2 (SEQ ID NO: 27), ending at 13 bpafter stop codon, GAC TGG CTG TGA CTG ATC TCT CT; primer 2 (SEQ ID NO:26), CTC ACT TAC ACT GCT GAA TGC AGA.

The primers for Rgh4 PCR are as follows:

Forward primer (SEQ ID NO: 48), ATG TCT CTC CCC AAA ACC CTA CTT TCT CTC;reverse primer (SEQ ID NO: 49), ending at 2 bp after stop codon, GGT TAACGG CAA TCC ATT GAA TCA AAG GAG.

PCR amplification is performed in an MJ Research PTC DNA Engine TMSystem, Model PTC-225 (MJ Research Inc, 590 Lincoln Street Waltham,Mass.). PCR is performed using the following components: 1 μl DNA, 5 μl10× buffer, 1 μl primer 1, 1 μl primer 2, 1 μl 10 mM dNTP, 1.5 μl 50 mMMgCl₂, 0.2 μl Taq. (Platinum), 39.3 μl H₂O. The PCR program used is asfollows: 95° C. for 10 minutes (step 1), 95° C. for 30 seconds (step 2),70° C. for 30 seconds/−1° C. per cycle/72° C. for 3 minutes (step 3),repeat steps two through three 9 times (step 4), 95° C. for 30 seconds(step 5), 60° C. for 30 seconds (step 6), 72° C. for 3 minutes (step 7),repeat steps five through seven 34 times (step 8), 4° C. forever (step9), end.

PCR products are separated on 1% agarose gel by electrophoresis. Asingle DNA band is excised from gel. Gel extraction is done usingCLONTECH NucleoSpin Extraction Kit (Clonetech Laboratories Inc., 1020East Meadow Circle, Palo Alto, Calif.). 2 μl of purified DNA is loadedon 1% agarose gel to check concentration. 40-100 ng of DNA is used forsubcloning.

A TOPO cloning reaction is done according to the following: 4 μl offresh PCR product, 1 μl Clontech Salt Solution, and 1 μl TOPO vector.The solution is mixed gently, incubated for 10 minutes at roomtemperature, and then placed on ice.

A one shot chemical transformation is performed as follows. 2 μl of theTOPO Cloning reaction is added to a vial of TOP 10 One Shot ChemicallyCompetent E. coli and mixed gently. The mixture is then Incubated on icefor 30 minutes. The cells are then heat-shocked for 30 seconds at 42°C., and immediately transferred to ice. 250 μl of SOC medium is thenadded, and the mixture is incubated at 37° C. for 1 hour. 80 pt is thenspread onto a selective plate, and 170 μl is spread onto another plate.The plates are incubated at 37° C. for 18-20 hours. The selective platesare LB agar plates with 100 μg/ml ampicillin, 40 μg/ml IPTG, and 40μg/ml X-GAL.

After incubation, 8-10 white or light blue colonies are selected. Thepositive colonies are inoculated into LB medium containing 50 μg/mlampicillin and incubated at 37° C. overnight. Sterilized glycerol isadded to make 15% glycerol stock, which can be stored at −80° C.

Sanger sequencing reactions are performed on subclones using BigDyeTerminators (Applied Biosystems, 850 Lincoln Centre Drive, Foster City,Calif.) and then analyzed on ABI 377/ABI 3700 automated sequencingmachines (Applied Biosystems, 850 Lincoln Centre Drive, Foster City,Calif.). The sequences are evaluated for quality and error probabilityusing the program, PHRED (Ewing and Green, Genome Res., 8:186-194(1998), Ewing et al., Genome Res., 8:175-185, (1998)), assembled usingthe phrap assembler and viewed using consed (Gordon et al., Genome Res.,8:195-202). An rhg1 candidate gene is found in BAC 240O17, and is about4.5 kb in size. An Rhg4 candidate was found in BAC 318013, and is about3.5 kb in size.

EXAMPLE 3

The physical mapping of a QTL (quantitative trait locus) is described inthis example. Mapping is initiated with linkage analysis of SSR (simplesequence repeats) markers. Markers that are shown to be linked to theQTL of interest are used to PCR screen the soy BAC library and identifycandidate BACs. Confirmed BACs are subcloned and sequenced, BAC-endsequenced, and fingerprinted. New markers are designed from good BAC-endsequences and used to screen the library, by either PCR or hybridizationto high density grid filters, in order to extend the contigs. A BAC-endsequence and fingerprint database of soy BACs is used in conjunctionwith the above methods to help build and extend contigs. Sequenced BACsare aligned, and overlapping BACs are placed into contigs. Thesecontigs, which contain unique sequences, are put into an ACEDB database,and predicted genes are annotated by hand using various programs.Candidates genes (for the gene of interest) are subcloned from genomicDNA of different lines by PCR using primers from outside the predictedcoding regions. These subclones are sequenced and screened for SNPs(single nucleotide polymorphisms) and INDELs (insertions/deletions), anddifferent haplotypes of the lines with and without the desired phenotypeare examined for correlations between the haplotype and phenotype.

A single trifoliate leaf is collected from the newest growth of fourweek old soybean plants. The leaf tissue is placed on ice and stored at−80° C. The frozen tissue is lyophilized and approximately 0.01 grams oftissue is used for DNA extraction. The leaf tissue is ground to powderin 1.4 ml tubes and 600 μl of DNA extraction buffer [0.5M NaCl, 0.1MTris-(hydroxymethyl) aminomethane pH 8.0, 0.05 Methylenediaminetetra-acetic acid (EDTA), 10.0 g L⁻¹ sodium dodecylsulfate (SDS), 2 g L⁻¹ phenantroline (dissolved in 0.01 L ethanol)]heated to 65° C. (with 0.77 g L⁻¹ dithiothreitol added immediatelybefore use) is added to each tube and mixed thoroughly. The samples areplaced in a 65° C. water bath for 15 minutes and shaken by hand after 10min. The samples are taken out of the water bath, cooled to roomtemperature, and 200 μl of 5 M KOAc is added to each tube. The samplesare inverted and placed at 4° C. for 20 min. Samples are thencentrifuged for 12 minutes at 6200×g and the supernatant (about 600 μl)is transferred to new tubes. DNA is precipitated with 330 μl of coldisopropanol and placed at −20° C. for 1 hr. The DNA is pelleted bycentrifuging at 6200×g for 10 minutes and is washed with 70% EtOH. TheDNA is pelleted by centrifugation at 6200×g for 10 minutes and driedusing a Speed-Vac. The DNA is dissolved in 100 μl of TE_(0.1) (0.01 MTris-HCl pH 8.0, 0.0001 M EDTA). The extraction yields 500 ng DNA μl⁻¹.

The polymerase chain reaction (PCR) is conducted with 5 to 10 ng genomicDNA in 10 μl volumes of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.001%gelatin, 1.5 mM MgCl₂, 0.1 mM of each dNTP, 150 nM of each primer, 0.01mM Cresol Red, 2% sucrose and 0.32 units of AmpliTaq DNA Polymerase(Perkin Elmer Instruments Inc., USA, 761 Main Avenue, Norwalk, Conn.).For thermocycling, the Gene Amp PCR System 9700 (Perkin ElmerInstruments Inc., USA, 761 Main Avenue, Norwalk, Conn.) is used with onestep of 94° C. for 3 min, then 32 cycles of 94° C., 47° C. and 72° C.steps of 25 sec each and one final step of 72° C. for 3 min. The PCRproducts are run on a 6% polyacrylamide gel (30 cm×8 cm×1 mm) in 1×TAE(40 MM Tris-dHC, pH 8.3, 1 mM EDTA) at 180 v for 45 min. The gels arestained using SYBR Gold (Molecular Probes, Eugene, Oreg.) permanufacturers instructions.

SSR primer screening for polymorphisms is performed using PIC, HS-1,Will and PI507354 genotypes. SSRs that are polymorphic and easy to score(i.e., Clear banding pattern and good separation between alleles) aremapped using the HS-1×PIC (F2) and/or Will x PI507354 (RIL) mappingpopulations. At least one SSR per BAC sequence is mapped. DNA markersthat exhibited codominant banding patterns are scored as homozygous forone or the other parent or as heterozygous, exhibiting both parentalalleles. Marker scores are checked for segregation distortion using thechi-squared test for goodness of fit to expected ratios. Linkagerelationships were determined using Mapmaker Version 3.0b with a LOD of3.0 (Whitehead Institute for Biomedical Research, Cambridge Mass.).

Thirty-two BAC DNA superpools (10 genomic equivalents) extracted fromeither 4608 clones (48 96-well microtiter plates) are used as templatesfor the first round of PCR screening. Following identification of thepositive superpools, the second screening is performed against 4-D BACDNA pools. Each clone of the superpool is addressed 4-dimentionally(7×7×12×8) and pooled in each dimension. Each set of 48 plates isdivided into 6 sets of 7 plates and one set of 6 plates, and partitionedin two ways. The first partition is in numerical order, plates 1-7,8-14, . . . 43-48 representing 7 group or stack pools. The secondpartition is according to plate position within each of the respectivestacks, plates [1, 8, 15, 22, 29, 36], [2, 9, 16, 23, 30, 37, 43] etc.,representing 7 plate pools. Each well of the 96-well plates contains 12columns and 8 rows. Clones from row 1 are pooled from all 48 plates togenerate the row 1 pool. Clones of rows 2, 3, 4 . . . 8, and columns 1,2, 3 . . . 12 are pooled to generate 8 row pools and 12 column poolsrespectively.

For each superpool, BAC DNA is extracted from a total of 34 subpools(7+7+8+12). Positive clones are identified by TaqMan/PCR screening ofthe 34 subpools if one positive clone is present. If more than onepositive clone is present in a superpool, a third round of screeningwith N4 PCR reactions is performed.

Addresses of candidate BACs are identified, and the candidates arestreaked out for single colony isolation and grown overnight at 37° C. Asingle, isolated colony is picked and streaked out and grown overnightat 37° C. PCR is repeated for the marker of interest (using the programdesigned for the relevant marker) using a smear of cells from the platestreaked from a single colony. The PCR product is run on a 2% agarosegel and purified using the Clonetech NucleoSpin Gel Extraction Kit(according to the manufacturer's instructions, Clonetech LaboratoriesInc., 1020 East Meadow Circle, Palo Alto, Calif.) and 10-50 ng of thepurified DNA are added to 10 pmol of each primer (forward and reverse),in a total volume of 6 μl of ddH₂O and 2 μl of BigDye Terminators(Applied Biosystems, 850 Lincoln Centre Drive, Foster City, Calif.). Thecycling conditions are: 96° C. for 1 minute (step 1), 96° C. for 10seconds (step 2), 50° C. for 5 seconds (step 3), 60° C. for 4 minutes(step 4), steps 2-4 are repeated for 24 cycles (step 5), and hold at 4°C.

The generated sequence is compared to the consensus sequence using DNAcomparison software. Confirmed clones are subcloned, sequenced, BAC-endsequenced, and Fingerprinted.

BAC-end sequencing is done using 3.2 pmol of SP6 and T7 primers(separately), approximately 600 ng-1 ug of BAC DNA (Autogen prepped,AutoGen Corp., 35 Loring Drive Framingham, Mass.) reaction, resuspendedin 6 μl of ddH₂O, and 4 μl of BigDye Terminators (Applied Biosystems 850Lincoln Centre Drive, Foster City, Calif.) to give a total reactionvolume of 10 ul. The cycling conditions are: 96° C. for 2 minutes (step1), 96° C. for 15 seconds (step 2), 50° C. for 15 seconds (step 3), 60°C. for 4 minutes (step 4), steps 2-4 are repeated for 50-60 cycles (step5), 72° C. for 2 minutes (step 6), hold at 4° C. or 10° C. (step 7).

The reactions are ethanol precipitated and loaded on capillarysequencers. The newly generated BAC-end sequence is trimmed from thevector sequence, and entered into a database containing approximately400,000 BAC-end sequences. Each BAC is blasted against the database tosearch for BAC-end matches extension of the contigs. New markers aredesigned from good BAC-end sequences, and these are then used torescreen the library in order to build up contigs across the region ofinterest. Screening can be done in either of two ways: as above (PCRstrategy), or by hybridization of high-density grid filters fromResearch Genetics (Research Genetics, 2130 Memorial Parkway, Huntsville,Ala.).

The probes used for hybridization are derived from clones or genomic DNAby PCR amplification using the vector or gene-specific primers, with theappropriate cycling conditions. PCR products are run on a 1% agarose gelcontaining ethidium bromide (0.2 ug/ml) in 1×TAE buffer at 100 volt for1-2 hrs. Isolated DNA fragments are excised and gel-purified using theClonetech NucleoSpin gel extraction kit (Clonetech Laboratories Inc.,1020 East Meadow Circle, Palo Alto, Calif.), before labeling. In orderto check the size of the fragments and concentration, 2 μl of eluted DNAplus loading buffer are loaded on a 1% agarose gel along with DNAmarkers of known concentration and size. All the probes used to screenthe library are tested individually for repetitiveness, with a smallerfilter spotted with random clones from the library along with somepositive control clones according to the protocol described below.

The A3244 soy library generated by a an EcoRI digest is spotted on 3high density grid filters from Research Genetics (Research Genetics,2130 Memorial Parkway, Huntsville, Ala.). Each filter has six fields,twelve 384 well plates are spotted in each field in duplicate, with atotal of 27,648 clones spotted on each filter. The plates are spotted ina 5×5 grid (12 clones per 5×5 grid) pattern within each field. Eachclone is spotted in duplicate with a specific orientation within the 5×5grid, which, together with the field position, gives information aboutits address. In a first round hybridization procedure, multiple probesare labeled separately and then pooled together to hybridize to BACfilters. Positive BACs identified in this procedure are deconvoluted byrehybridization with the individual probes.

A hybridization oven is set at 65° C., and Church Buffer (0.5 M SodiumPhosphate, pH 7.0, 7% SDS, 1% bovine serum albumin, 1 mM EDTA, 100 μg/mlsalmon sperm DNA) is prewarmed to 65° C. Membranes are washed in 500 mlof 0.1×SSC, 0.1% SDS in a large container at room temperature for 5minutes with gentle shaking (50 rpm) on a rotary shaker. The membranesare rinsed with 500 ml of 0.1×SSC (no SDS) for 1 minute. The washsolution is poured off, and 500 ml of 6×SSC (no SDS) is added toequilibrate the membranes. Three filters are placed in a tube. Thefilters are separated from each other and the sides of the tube by alayer of mesh. Each tube is filled with 6×SSC and shaken gently with thetube vertical to help eliminate bubbles between the filters and tubewall. The 6×SSC solution is poured off, and 25 ml of pre-warmed Churchbuffer is added. The bottles are rotated in a hybridization oven at 60rpm and 65° C. for 30 minutes or longer.

Probes are labeled using 1 μl of 40-50 uCi/μl [α³²P dCTP], 50 ng ofpurified DNA in 49 μl of ddH₂O, and Read-To-Go Labeling Beads fromAmersham Pharmacia according to the manufacturers instructions (AmershamPharmacia, Uppsala, Sweden). The probes are purified using the Bio-SpinColumn P30 from BioRad according to manufacturers instructions (Bio-RadLaboratories, 3316 Spring Garden Street, Philadelphia, Pa.). To 1 μl ofthe column-purified probe is added to a minipoly-Q vial (liquidscintillation vial) for each probe. 5 ml of scintillation liquid isadded to each vial, and radiation activity for each vial is measuredusing a liquid scintillation counter.

After the probes are purified and counted for radioactivity, 10-20probes and one control probe (from 50 μl reaction) are pooled with 10⁷cpm/probe each, into one 1.5 ml eppendorf tube. The pooled probes aredenatured at 99° C. in a sand heating block for 10 minutes. The tubesare cooled on ice or ice water about 2 minutes, and then spun down at14,000 rpm for 30 seconds in microcentrifuge. The tubes arepre-hybridized in 25 ml of Church buffer for at least 30 minutes, whichis then poured off. 40 ml of fresh hybridization solution (pre-warmedChurch buffer) is added. The pooled-probe solution is added to thehybridization tube. The tube is rotated in the hybridization oven at 60rpm, 65° C. overnight.

The probe solution is poured off, 30 ml of pre-warmed (65° C.) 1×SSC,0.1% SDS washing solution is added to the hybridization tube, thehybridization tube is rotated in the hybridization oven (at 65° C.) for15 minutes, and the process is repeat two times. At the last wash, thetube is rotated 180° and at the same speed for 15 minutes at 65° C. Thewashing solution is poured off, and 2×SSC (no SDS) is added.

Excess liquid is removed from each filter by placing the filter on apiece of 3 MM paper. The washed filter is placed on developed film withthe DNA-side up (the side BACs were spotted on), covered with Saranwrap, and squeezed to force out liquid and bubbles. The Saran wrap isfolded to the other side of the film, fixed it with tape, and then driedKimwipes. The wrapped filters are placed into a film cassette with theDNA-side up (the side BACs were spotted on), which is placed on BioMaxMS film (Biomax Technologies Inc., Vancouver, BC, Canada) in a darkroom,and exposed overnight at room temperature without an intensifyingscreen. Film is developed with a film developer in the dark room thenext day, and each film is labeled with filter number, probe used forhybridization, exposure time, and date.

Starting from Field 3, a 384-well grid is put on the field with the A1position of the grid on the upper right, and the grid is aligned to theimage. The row and column position for each positive clone on the BACrecording spreadsheet is determined and recorded. The pattern of thehybridization signal is matched to known patterns. There are 6 platereference numbers for each of twelve patterns, which are arranged in thesame manner as the 6 fields. Based on the signal pattern and fieldnumber, a plate reference number is determined for each positive clone.The grid is moved to the next field and the process is repeated. Theoriginal plate number (P) is determined using the following formula:P=(N−1)×72+R, where N is the filter number on which the identified cloneis present and R is the plate reference number previously determined.The complete address of the identified clone is given by the originalplate number plus its position on the plate determined previously. BACs'addresses are identified and converted to “imp” files according to aQ-bot file format.

24 working plates are loaded into a Q-bot (Genetix, Queensway, NewMilton, Hampshire, United Kingdom) 6-high hotel and media-filled 96-wellplates are placed on the deck. The Q-bot is run following the standardmanual using the program called “Rearraying98” with the settings givenin Appendix III of the accompanying manual: BAC-Picking. Platescontaining picked clones are placed in a shaker incubator and grownovernight at 37° C. at 200 rpm.

35 μl DNA solution are transferred from 96-well plates into a 384-wellplate using a Platemate such that 4 96-well plates of DNA are combinedinto one 384-well plate. The 384-pin head (puck) is washed in 10% SDSsolution for 5 minutes, ultrasonicated in a water bath for 3 minutes,washed with 70% ethanol for 1 min., and air dried for 3 minutes. The384-well DNA source plates and membranes are arranged on the deckaccording to the instruction from the manual and the spotted grid designchosen for the membrane. Spotting pattern are designed so that there isone control probe at each of the 4 corners of the membrane. Anasymmetric pattern is used to orient filters. The control probeconcentration is about 5 ng/ul. Zeus is run according to instructions.If the DNA concentration is lower than 5 ng/ul, the Zeus is run a secondtime to double the amount of spotted DNA on the membrane. One of theempty spots is spot dyed, if available, using one 384-well dye plate. Ifan empty spot is not available, it is printed on one of the DNA spots.This spot marks the position for cutting filters into small membranes(9×12 cm). Membranes are interleaved between 3M papers and left toair-dry. Each corner of each membrane is marked with a permanent markerand numbered. Filters are denatured on the surface of 3M paper soakedwith denaturalization solution for 4 minutes, and then neutralized onthe surface of 3 M paper soaked with neutralization solution for 5minutes. The filters are washed with 2×SSC for 5 minutes and then airdried. The filters are then baked at 80° C. for 1 hr. and cut intoindividual small membranes (9×12 cm) according to the marked corner.

To confirm and deconvolute, hybridizations are done as before, but withnewly generated filters, and each probe is done separately with a singlefilter using the smaller tube. 15 ml of Church buffer is used for thehybridization.

Fingerprints are generated by digesting the BAC DNA with Hind III for 3hours at 37° C. and running the reaction on a 0.8% gel at 200V for 19hours. The gels are stained with SybrGreen, while shaking at roomtemperature for 45 minutes, and scanned with a Flourimager. The bandsare sized using Frag software and the fingerprints are assembled intocontigs within FPC. Every time new clones are added the contigs arerebuilt using a tolerance of 10 and a cutoff of 10⁻⁹.

Subclones are generated and Sanger sequencing reactions were performedon randomly chosen subclones using BigDye Terminators (AppliedBiosystems, 850 Lincoln Centre Drive, Foster City, Calif.) then analyzedon ABI 377/ABI 3700 automated sequencing machines (Applied Biosystems,850 Lincoln Centre Drive, Foster City, Calif. 7-8 fold sequence coverageis thereby generated across the BAC. The sequences are evaluated forquality and error probability using the program, phred, assembled usingthe phrap assembler, and viewed using consed, as in example 2. ForBermuda standard BACs, all contigs are ordered and oriented and all gapsare closed using a directed primer walking strategy. A final qualityvalue of phred40 (1 base error in 10,000 bases) with no gap regions,double coverage or two chemistries across single stranded areas isachieved.

The sequence contigs are put into an ACEDB database along with soy ESTand plant EST matches, along with Blastx, Tblastx, and Plant Blastnhits. Genemark.hmm is used to predict possible genes, and GeneFinder isused to predict splicing sites, ORFs, potential coding regions, as wellas start and stop codons. The contigs are then annotated by hand andpredicted genes accepted, edited, and modified based on thecharacteristics present in the sequence and matches to protein,nucleotide, and EST databases.

The high-density BAC library membranes used for hybridization are madeby Research Genetics (Research Genetics, 2130 Memorial Parkway,Huntsville, Ala., using a modified Q-bot (Genetix, Queensway, NewMilton, Hampshire, United Kingdom), 384-well plates containing BACs arespotted onto 22 cm×22 cm Hybond N+ membranes (Amersham Pharmacia,Uppsala, Sweden). Bacteria from 72 plates are spotted twice onto onemembrane, giving 55,296 colonies in total, or 27,648 unique clones permembrane. The plates are spotted into six “fields” per membrane, witheach field having 12 plates spotted in duplicate. This spotting formatresults in six fields with 384 grids in each field. Each grid is a 5×5matrix containing 12 unique clones in duplicate, with the centerposition left empty. The two positions occupied by each clone induplicate are designed to give a unique pattern that indicates the platelocation of each clone. After spotting, the bacteria on the membrane areincubated for 8 hours on LB-agar plates containing 12.5 ug/mlchloramphenicol. The membranes are then denatured, neutralized, washedin a standard procedure, UV-light crosslinked, and air-dried. Themembranes can be stored and shipped at room temperature.

Every reference, patent, or other published work cited above is hereinincorporated by reference in its entirety.

1-72. (canceled)
 73. A method of introgressing an allele into a soybeanplant comprising (A) crossing at least one SCN resistant soybean plantwith at least one SCN sensitive soybean plant in order to form asegregating population, (B) screening said segregating population withone or more nucleic acid markers to determine if one or more soybeanplants from said segregating population contains an rhg1 SCN resistantallele and an Rhg4 SCN resistant allele, wherein said rhg1 SCN resistantallele is an allele having a deletion of 19 nucleotides corresponding toSEQ ID NO: 2 and encompassing position 48881, and said Rhg4 SCNresistant allele is an allele having one or more polymorphisms at aposition in SEQ ID NO: 4 selected from the group consisting of 111933,112065, 112101, and 112461, and (C) selecting, if present, one or moresoybean plants of said segregating population containing said deletionand said one or more polymorphisms located at a position in SEQ ID NO:4.
 74. The method according to claim 73, wherein said one or moresoybean plants of said segregating population have a yellow soybeanseed.
 75. A method of introgressing an allele into a soybean plantcomprising (A) crossing at least one SCN resistant soybean plant with atleast one SCN sensitive soybean plant in order to form a segregatingpopulation, (B) screening said segregating population with one or morenucleic acid markers to determine if one or more soybean plants fromsaid segregating population contains an rhg1 SCN resistant allele and anRhg4 SCN resistant allele, wherein said rhg1 SCN resistant allele is anallele having one or more first polymorphisms located at a position inSEQ ID NO: 2 selected from the group consisting of 45173, 45309, 47057,47140, 47208, 47571, 47617, 47796, 47856, 47937, 48012, 48060, 48073,48135, 48279, 48413, 48681, 48881, 49012, and 493161, and said Rhg4 SCNresistant allele is an allele having one or more second polymorphisms ata position in SEQ ID NO: 4 selected from the group consisting of 111933,112065, 112101, and 112461, and (C) selecting, if present, one or moresoybean plants of said segregating population containing said one ormore first polymorphisms and said one or more second polymorphisms. 76.The method according to claim 75, wherein said one or more soybeanplants of said segregating population have a yellow soybean seed.
 77. Amethod of introgressing an allele into a soybean plant comprising (A)crossing at least one SCN resistant soybean plant with at least one SCNsensitive soybean plant in order to form a segregating population, (B)screening said segregating population with one or more nucleic acidmarkers to determine if one or more soybean plants from said segregatingpopulation contains an rhg1 SCN resistant allele and an Rhg4 SCNresistant allele, wherein said rhg1 SCN resistant allele is an allelehaving one or more first polymorphisms in a protein coding regioncorresponding to nucleotides 45163 to 45314, 45450 to 45509, 46941 to48763 or 48975 to 49573 of SEQ ID NO: 2, and said Rhg4 SCN resistantallele is an allele having one or more second polymorphisms in a proteincoding region corresponding to nucleotides 111805 to 113968 or 114684 to115204 of SEQ ID NO: 4, and (C) selecting, if present, one or moresoybean plants of said segregating population containing said one ormore first polymorphisms and said one or more second polymorphisms. 78.The method according to claim 77, wherein said one or more soybeanplants of said segregating population have a yellow soybean seed. 79.The method according to claim 77, wherein said one or more firstpolymorphisms and said one or more second polymorphisms are singlenucleotide polymorphisms.
 80. The method according to claim 77, whereinsaid one or more first polymorphisms and said one or more secondpolymorphisms are INDEL or simple sequence repeat (SSR) polymorphisms.81. The method according to claim 77, wherein said one or more firstpolymorphisms are selected from the group consisting of 45173, 45309,47057, 47140, 47208, 47571, 47617, 47796, 47856, 47937, 48012, 48060,48073, 48135, 48279, 48413, 48681, 49012, and 49316 of SEQ ID NO:
 2. 82.The method according to claim 77, wherein said one or more secondpolymorphisms are selected from the group consisting of 111933, 112065,112101, and 112461 of SEQ ID NO:
 4. 83. A method of introgressing one ormore alleles comprising (A) crossing at least one SCN resistant soybeanplant with at least one SCN sensitive soybean plant in order to form asegregating population, (B) screening said segregating population withone or more nucleic acid markers to determine if one or more soybeanplants from said segregating population contains an rhg1 SCN resistantallele and an Rhg4 SCN resistant allele, wherein said rhg1 SCN resistantallele is an allele having one or more first polymorphisms in a proteincoding region corresponding to nucleotides 45163 to 49573 of SEQ ID NO:2, and said Rhg4 SCN resistant allele is an allele having one or moresecond polymorphisms in a protein coding region corresponding tonucleotides 111805 to 115204 of SEQ ID NO: 4, and (D) selecting one ormore soybean plants of said segregating population having said rhg1 SCNresistant allele and said Rhg4 resistant allele.
 84. The methodaccording to claim 83, wherein said one or more soybean plants of saidsegregating population have a yellow soybean seed.
 85. The methodaccording to claim 83, wherein said one or more first polymorphisms andsaid one or more second polymorphisms are single nucleotidepolymorphisms.
 86. The method according to claim 83, wherein said one ormore first polymorphisms and said one or more second polymorphisms areINDEL or simple sequence repeat (SSR) polymorphisms.
 87. The methodaccording to claim 83, wherein said rhg1 SCN resistant allele is anallele having one or more first polymorphisms in a protein coding regioncorresponding to nucleotides 45163 to 45314, 45450 to 45509, 46941 to48763 or 48975 to 49573 of SEQ ID NO:
 2. 88. The method according toclaim 83, wherein said one or more first polymorphisms are selected fromthe group consisting of 45173, 45309, 47057, 47140, 47208, 47571, 47617,47796, 47856, 47937, 48012, 48060, 48073, 48135, 48279, 48413, 48681,49012, and 49316 of SEQ ID NO:
 2. 89. The method according to claim 83,wherein said rhg1 SCN resistant allele is an allele having a deletion of19 nucleotides corresponding to SEQ ID NO: 2 and encompassing position48881.
 90. The method according to claim 83, wherein said Rhg4 SCNresistant allele is an allele having one or more second polymorphisms ina protein coding region corresponding to nucleotides 111805 to 113968 or114684 to 115204 of SEQ ID NO: 4
 91. The method according to claim 83,wherein said one or more second polymorphisms are selected from thegroup consisting of 111933, 112065, 112101, and 112461 of SEQ ID NO: 4.